PNA monomer and precursor

ABSTRACT

This application relates to monomers of the general formula (I) for the preparation of PNA (peptide nucleic acid) oligomers and provides method for the synthesis of both predefined sequence PNA oligomers and random sequence PNA oligomers:  
                 
wherein E is nitrogen or C—R′; J is sulfur or oxygen; R′, R1, R2, R3, R4 is independently H, halogen, alkyl, nitro, nitrile, alkoxy, halogenated alkyl, halogenated alkoxy, phenyl or halogenated phenyl, R5 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases in the presence of thiol.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to monomers suitable for the preparationof PNA oligomers. The present invention also relates to precursors tothe monomers and methods of making the PNA monomers from the precursors.Further, the invention relates to methods of making PNA oligomers usingthe PNA monomers.

2. General Background and State of the Art

In the last two decades, attempts to optimize the properties ofoligonucleotide by modification of the phosphate group, the ribose ring,or the nucleobase have resulted in a lot of discoveries of newoligonucleotide derivatives for the application in the fields of DNAdiagnostics, therapeutics in the form of antisense and antigene, and thebasic research of molecular biology and biotechnology (U. Englisch andD. H. Gauss, Angew. Chem. Int. Ed. Engl. 1991, 30, 613-629; A. D.Mesmaeker et al. Curr. Opinion Struct. Biol. 1995, 5, 343-355; P. E.Nielsen, Curr. Opin. Biotech., 2001, 12, 16-20.). The most remarkablediscovery is peptide nucleic acid which was reported by the Danish groupof Nielsen, Egholm, Buchardt, and Berg (P. E. Nielsen et al., Science,1991, 254, 1497-1500). PNA is DNA analogue in which anN-(2-aminoethyl)glycine polyamide replaces the phosphate-ribose ringbackbone, and methylene-carbonyl linker connects natural as well asunnatural nucleo-bases to central amine of N-(2-aminoethyl)glycine.Despite radical change to the natural structure, PNA is capable ofsequence specific binding to DNA as well as RNA obeying the Watson-Crickbase pairing rule. PNAs bind with higher affinity to complementarynucleic acids than their natural counterparts, partly due to the lack ofnegative charge on backbone, a consequently reduced charge-chargerepulsion, and favorable geometrical factors (S. K. Kim et al., J. Am.Chem. Soc., 1993, 115, 6477-6481; B. Hyrup et al., J. Am. Chem. Soc.,1994, 116, 7964-7970; M. Egholm et al., Nature, 1993, 365, 566-568; K.L. Dueholm et al., New J. Chem., 1997, 21, 19-31; p. Wittung et al., J.Am. Chem. Soc., 1996, 118, 7049-7054; M. Leijon et al., Biochemistry,1994, 9820-9825.). Also it was demonstrated that the thermal stabilityof the resulting PNA/DNA duplex is independent of the salt concentrationin the hybridization solution (H. Orum et al., BioTechniques, 1995, 19,472-480; S. Tomac et al., J. Am. Chem. Soc., 1996, 118, 5544-5552.). AndPNAs can bind in either parallel or antiparallel fashion, withantiparallel mode being preferred (E. Uhlman et al., Angew. Chem. Int.Ed. Engl., 1996, 35, 2632-2635.).

A mismatch in a PNA/DNA duplex is much more destabilizing than amismatch in a DNA/DNA duplex. A single base mismatch results in 15° C.and 11° C. lowering of the Tm of PNA/DNA and DNA/DNA, respectively.Homopyrimidine PNA oligomers and PNA oligomers with a highpyrimidine/purine ratio can bind to complementary DNA forming unusuallystable PNA2/DNA triple helices (P. E. Nielsen et al., Science, 1991,254, 1497-1500; L. Betts et al., Science, 1995, 270, 1838-1841; H.Knudsen et al., Nucleic Acids Res., 1996, 24, 494-500.). Although PNAshave amide bonds and nucleobases, PNAs show great resistance to bothnuclease and protease. In contrast to DNA, which depurinates ontreatment with strong acids and hydrolyses in alkali hydroxides, PNAsare completely acid stable and sufficiently stable to weak bases.

Generally, PNA oligomers are synthesized using the well establishedsolid phase peptide synthesis protocol. New strategies for monomers havebeen developed independently by several groups to optimize PNA oligomersynthesis. The preparation of PNA monomers can be divided into thesynthesis of a suitably protected N-aminoethylglycine and a suitablyprotected nucleobase acetic acid derivatives, which is followed bycoupling both.

The first synthetic strategy reported for PNA oligomer synthesis wasMerrifield solid phase synthesis using t-Boc/benzyloxycarbonylprotecting group strategy wherein the backbone amino group protectedwith the t-Boc and the exocyclic amino groups of the nucleobases areprotected with the benzyloxycarbonyl (P. E. Nielsen et al., Science,1991, 254, 1497-1500; M. Egholm et al., J. Am. Chem. Soc., 1992, 114,9677-9678; M. Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895-1897; M.Egholm et al., J. Chem. Soc. Chem. Commun., 1993, 800-801; K. L. Dueholmet al., J. Org. Chem., 1994, 59, 5767-5773; WO 92/20702). PNA monomersprotected with t-Boc/benzyloxycarbonyl are now commercially availablebut are inconvenient to use because repeated treatment with TFA isrequired for t-Boc deprotection and the harsh HF ortrifluoromethanesulfonic acid treatment required for cleavage from theresin and deprotection of benzyloxycarbonyl group from exocyclic amineof nucleobases. Thus, this strategy is not compatible with the synthesisof many types of modified PNA oligomers such as PNA-DNA chimera.Furthermore, the use of hazardous acids, such as HF ortrifluoromethanesulfonic acid, is not commercially embraced in view ofsafety concerns for the operator and the corrosive effect on automationequipment and lines. In addition, the t-Boc/benzyloxycarbonyl protectionstrategy is differential strategy which is defined as a system ofprotecting groups wherein the protecting groups, are removed by the sametype of reagent or condition, but rely on the different relative ratesof reaction to remove one group over the other. For example, in thet-Boc/benzyloxycarbonyl protecting strategy, both protecting groups areacid labile, but benzyloxycarbonyl group requires a stronger acid forefficient removal. When acid is used to completely remove the more acidlabile t-Boc group, there is a potential that a percentage ofbenzyloxycarbonyl group will also be removed contemporaneously.Unfortunately, the t-Boc group must be removed from amino group ofbackbone during each synthetic cycle for the synthesis of oligomer. ThusTFA is strong enough to prematurely deprotect a percentage of the sidechain benzyloxycarbonyl group, thereby introducing the possibility ofoligomer branching and reducing the overall yield of desired product.

In another effort to find a milder deprotecting method for PNA oligomersynthesis that would be compatible with DNA oligomer synthesis, severalresearch groups have developed PNA monomers protected with Mmt/acylwherein the backbone amino group protected with the Mmt and theexocyclic amino groups of the nucleobases are protected with an acylgroup such as benzoyl, anisoyl, and t-butyl benzoyl for cytosine andadenine, or isobutyryl, acetyl for guanine (D. W. Will et al.,Tetrahedron, 1995, 51, 12069-12082; P. J. Finn et al., Nucleic AcidResearch, 1996, 24, 3357-3363; D. A. Stetsenko et al., Tetrahedron Lett.1996, 3571-3574; G. Breipohl et al., Tetrahedron, 1997, 14671-14686.).

Alternative PNA monomers protected with Fmoc/benzhydryloxycarbonyl arealso commercially available wherein the backbone amino group protectedwith the Fmoc and the exocyclic amino groups of the nucleobases areprotected with the benzhydryloxycarbonyl (J. M. Coull, et al., U.S. Pat.No. 6,133,444). But Fmoc/benzhydryloxycarbonyl strategy has severaldrawbacks such as side reaction during the Fmoc deprotection process andinstability of monomer in solution. The most critical side reaction isthe migration of the nucleobase acetyl group from the secondary aminofunction to the free N-terminal amino function of aminoethylglycinebackbone under Fmoc deprotection condition (L. Christensen et al., J.Pept. Sci. 1995, 1,175-183). The N-acetyl transfer reactions in everycycles during oligomer synthesis result in accumulation of side productswhich are hard to separate due to similar polarity and same molecularweight. Also the Fmoc protecting group is very unstable in the presenceof trace amine. Thus, the selection of the solvent for the PNA monomersshould be cautious. Generally, N-methylpyrrolidone of high quality isrecommended. This requires higher cost in the synthesis of PNA oligomer.

The synthesis of PNA oligomers using Fmoc/benzyloxycarbonyl (S. A.Thomson et al., Tetrahedron, 1995, 6179-6194.) and Fmoc/Mmt (G. Breipohlet al., Bioorg. Med. Chem. Lett., 1996, 6, 665-670.) protected monomerhas also been reported. However, all of these methods have seriousdrawbacks in terms of monomer solubility and preparation, harsh reactioncondition, and side reactions either during monomer synthesis and/or PNAoligomer synthesis.

In other efforts to find new monomers, cyclic monomers were reported byISIS and Biocept. The first strategy developed by ISIS replacesprotected backbone by morpholinone (U.S. Pat. No. 5,539,083), but thestrategy has serious drawback in that the hydroxy functional groupgenerated by coupling reaction should be converted to amine functionalgroup in every elongation step during oligomer synthesis. Alternatively,the protected aminoethylglycine part is replaced by N-t-Boc-piperazinone(WO 00/02899). However, this strategy also has several drawbacks interms of monomer reactivity in oligomerization and the same problems asseen in linear t-Boc strategy as described above.

Despite recent advances, there remains a need for new monomer thatincreases yield, lowers synthetic cost, and is suitable for automaticand parallel synthesis.

SUMMARY OF THE INVENTION

The present invention provides novel monomers for increased efficiency,high yield, and convenience during synthesis of PNA oligomers. Anotherobject is to provide PNA monomers that can be conveniently applied toinstrumentation such as automated synthesizer for synthesis of PNAoligomers. The novel monomers according to the present invention arecompounds having general formula I:

-   -   wherein    -   E may be nitrogen or C—R′,    -   J may be sulfur or oxygen,    -   R′, R1, R2, R3, R4 may be independently H, halogen such as F,        Cl, Br or I, CF₃, alkyl; preferably C₁-C₄ alkyl, nitro, nitrile,        alkoxy, preferably C₁-C₄ alkoxy, halogenated (such as F, Cl, Br        and I) alkyl, preferably halogenated C₁-C₄ alkyl, or halogenated        (such as F, Cl, Br and I) alkoxy, preferably halogenated C₁-C₄        alkoxy, phenyl, or halogenated (such as F, Cl, Br and I) phenyl,    -   R5 may be H or protected or unprotected side chain of natural or        unnatural α-amino acid, and    -   B may be a natural or unnatural nucleobase, wherein when said        nucleobase has an exocyclic amino function, said function is        protected by protecting group which is labile to acids but        stable to weak to medium bases in the presence of thiol. In        another embodiment, B may be thymine (T), cytosine (C), adenine        (A), or guanine (G).

Further in particular, the protecting group for the exocyclic aminofunction of B has a general formula:

The group represented by R7 may have a general formula:

The residue represented by Y₁-Y₁₀ is independently selected fromhydrogen, halogen, such as F, Cl, Br, alkyl, preferably methyl, ethyl,t-butyl, and alkoxy, such as methoxy, ethoxy, and t-butyloxy.

In another embodiment, R7 may have a general formula;

The residue represented by Z₁-Z₅ is independently selected fromhydrogen, halogen, such as F, Cl, Br, alkyl, preferably methyl, ethyl,t-butyl, and alkoxy, such as methoxy, ethoxy, and t-butyloxy, andmethylene dioxy of adjacent two residues.

In another embodiment, R7 may have a general formula;

The residue represented by R8 may be alkyl or phenyl.

Further in particular, the protecting group of B may bebenzyloxycarbonyl, benzhydryloxycarbonyl, 4-methoxybenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, piperonyloxycarbonyl, or2-methylthioethoxycarbonyl.

The invention is also directed to nucleobase B, which may be protectedby piperonyloxycarbonyl derivatives.

In particular, the invention is directed to a cytosine moiety that isprotected by piperonyloxycarbonyl derivatives having a general formula:

The residue represented by Q₁-Q₅ may be independently selected fromhydrogen, halogen, such as F, Cl, Br, and I, nitro, alkyl, such asmethyl, ethyl, and t-butyl, and alkoxy, such as methoxy, ethoxy, andt-butyloxy.

The invention is also directed to an adenine moiety that is protected bypiperonyloxycarbonyl derivatives having a general formula:

-   -   wherein    -   Q₁, Q₂, Q₃, Q₄ and Q₅ are as defined above.

The invention is also directed to a guanine moiety that is protected bypiperonyloxycarbonyl derivatives having a general formula:

-   -   wherein    -   Q₁, Q₂, Q₃, Q₄ and Q₅ are as defined above.

The present invention further provides for methods of preparingcompounds of general formula I from compounds of general formula V.

-   -   wherein    -   E, J, R1, R2, R3, R4, R5 and B are as defined above,    -   the protecting group of B when said nucleobase of B has an        exocyclic amino function is also as described above, and    -   R6 may be H, alkyl, preferably (C₁-C₄) alkyl (such as methyl,        ethyl, and t-butyl), or aryl.

In another embodiment, the invention provides for compounds of generalformula V and their preparation methods from compounds of generalformula II.

-   -   wherein    -   E, J, R1, R2, R3, R4, R5, and R6 are as defined above.

Also, the present invention provides compounds of formula II and theirpreparation methods.

In another embodiment, the invention is directed to a method of makingthe compound of formula V, comprising coupling reaction of a compound offormula II with a nucleobase acetic acid moiety in the presence ofnon-nucleophilic organic base and a coupling reagent that is customarilyused in peptide synthesis.

The present invention further provides methods of preparing compounds offormula I from compounds of general formula IV.

-   -   wherein    -   E, J, R1, R2, R3, R4, and R5 are as defined above, and    -   HX is organic or inorganic acid.

The present invention also provides compounds of general formula IV andtheir free acid form, and their preparation methods.

The present invention further provides methods of preparing compounds ofgeneral formula IV from compounds of general formula II.

The invention is also directed to a method of making the compound offormula I, comprising coupling reaction of a compound of formula IV witha nucleobase acetic acid moiety in the presence of non-nucleophilicorganic base and a coupling reagent that is customarily used in peptidesynthesis.

The invention is also directed to a method of making the compound offormula I, comprising cyclizing a compound of formula VI in the presenceof a coupling reagent that is customarily used in peptide synthesis ormixed anhydride.

The entities represented by E, J, R1, R2, R3, R4, R5, and B are asdefined above. The protecting group of B when said nucleobase of B hasan exocyclic amino function is also as set forth above.

The invention is directed to a compound of formula II, for which itsresidues are defined as above. In particular, the R5 residue may be H orprotected or unprotected side chain of natural α-amino acid. In otherembodiments, the compound of formula II may have the followingconfiguration: R1, R2, R3, and R4 may be H, E is nitrogen, and J issulfur. In other embodiments, R1, R3, and R4 may be H, R2 may be Cl, Eis nitrogen, and J is sulfur. In other embodiments, R3 and R4 may be H,R1 may be Cl, R2 may be methoxy, E is nitrogen, and J is sulfur.

The invention is also directed to a method of making the compound offormula II, comprising reacting benzothiazole-2-sulfonylchloride,benzoxazole-2-sulfonylchloride benzo[b]thiophene-2-sulfonylchloride, orbenzofuran-2-sulfonylchloride, derivative with 2-aminoethylglycine esterin the presence of non-nucleophilic organic base. The invention isfurther directed to a compound having formula IV and its free acid form.The residues for formula IV are as defined above. In particular, the R5residue may be H or protected or unprotected side chain of naturalα-amino acid. In other embodiments, the compound of formula IV may havethe following configuration: R1, R2, R3, and R4 may be H; E is nitrogenand 0.1 is sulfur. In other embodiments, R1, R3, and R4 may be H, R2 maybe Cl, E is nitrogen, and J is sulfur. In other embodiments, R3 and R4may be H, R1 may be Cl, R2 may be methoxy, E is nitrogen, and J issulfur.

The invention is further directed to a method of making the compound offormula IV, comprising cyclizing a compound of formula III in thepresence of a coupling reagent that is customarily used in peptidesynthesis or mixed anhydride, followed by deprotection of a protectiongroup such as t-Boc in acid.

The entities represented by E, J, R1, R2, R3, R4, and R5 are as definedabove.

The invention is also directed to a method of making PNA oligomer,comprising linking together the compound of formula I.

It is to be understood that the “R” groups, and E and J designationscited above apply to all of the compounds of formulae I-VI. It is alsoto be understood that the designations also apply to the compounds asthey undergo the processes of the invention.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto. It is further to beunderstood that the chemical formulae as labeled in the figures serve asrepresentative embodiments. The inventive chemical formulae, arediscussed throughout the specification. The recited chemical formulae inthe figures are not meant to limit the scope of the recited compounds inany way.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIG. 1 shows a chart of the chemical structures of naturally andnon-naturally occurring nucleobases useful for DNA recognition.

FIG. 2 shows another chart of the chemical structures of naturally andnon-naturally occurring nucleobases useful for DNA recognition.

FIG. 3 shows a schematic representation of the synthesis of protectedbackbone.

FIG. 4 shows a schematic representation of the synthesis of protectedpiperazinone as a precursor for monomer.

FIG. 5 shows a schematic representation of the synthesis of PNA monomer.

FIG. 6 shows a schematic representation of the alternative synthesis ofPNA monomer.

FIG. 7 shows schemes of preparation of nucleobases protected withpiperonyloxycarbonyl.

FIG. 8 shows a schematic representation of the synthesis of PNA thyminemonomer.

FIG. 9 shows a schematic representation of the alternative synthesis ofPNA thymine monomer.

FIG. 10 shows a schematic representation of the synthesis of PNAcytosine monomer

FIG. 11 shows a schematic representation of the alternative synthesis ofPNA cytosine monomer.

FIG. 12 shows a schematic representation of the synthesis of PNA adeninemonomer.

FIG. 13 shows a schematic representation of the alternative synthesis ofPNA adenine monomer.

FIG. 14 shows a schematic representation of the synthesis of PNA guaninemonomer.

FIG. 15 shows a schematic representation of the alternative synthesis ofPNA guanine monomer.

FIG. 16 shows a schematic representation of the PNA oligomer synthesisfrom PNA monomers.

FIG. 17 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 1

FIG. 18 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 2

FIG. 19 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 3

FIG. 20 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 4

FIG. 21 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 5

FIG. 22 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 6

FIG. 23 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 7

FIG. 24 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 8

FIG. 25 shows HPLC and MALDI-TOF results for synthesized oligomer SEQ IDNO: 9

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that the formulae depicted in the drawings aremerely exemplified versions, and where a particular formula is referredto in the claims, the version discussed in the text of the specificationis meant.

In the present invention, benzothiazole-2-sulfonyl,benzo[b]thiophene-2-sulfonyl, benzoxazole-2-sulfonyl, orbenzofuran-2-sulfonyl group of compounds having general formula I playan important role not only as protecting groups of amine of backbone butalso as activating groups for coupling reaction. The monomers havingdescribed characteristics are useful for the synthesis of PNA oligomersby manual or automated synthesizer and the preparation of PNA, oligomerlibrary by combinatorial chemistry. Nucleobase B in the general formulaI is naturally attached at the position found in nature, i.e., positionI for thymine or cytosine, and position 9 for adenine or guanine, aswell as for non-naturally occurring nucleobase (nucleobase analog), ornucleobase binding moiety. Some nucleobases and illustrative syntheticnucleobases are shown in FIG. 1 and FIG. 2.

Preparation of Protected Backbones

The first step for the preparation of novel monomers having generalformula I is synthesis of N-[2-(benzothiazole, benzoxazole,benzo[b]thiophene, or benzofuran-2-sulfonylamino)-ethyl]-glycinederivatives having the formula II:

-   -   wherein E is nitrogen and J is sulfur for        N-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine derivatives;        E is nitrogen and J is oxygen for        N-[2-(benzoxazole-2-sulfonylamino)-ethyl]-glycine derivatives; E        is C—R′ and J is sulfur for        N-[2-(benzo[b]thiophene-2-sulfonylamino)-ethyl]-glycine        derivatives; E is C—R′ and J is oxygen for        N-[2-(benzofuran-2-sulfonylamnio)-ethyl]-glycine derivatives;        and R′, R1, R2, R3, R4, R5, and R6 are as defined above.

Derivatives having formula II are generally synthesized by selectivereaction of primary amine of 2-aminoglycine derivatives with sulfonylchloride compounds having the general formula:

-   -   wherein E is nitrogen and J is sulfur for        benzothiazole-2-sulfonyl chloride derivatives; E is nitrogen and        J is oxigen for benzoxazole-2-sulfonyl chloride derivatives; E        is C—R′ and J is sulfur for benzo[b]thiophene-2-sulfonyl        chloride derivatives; E is CH and J is oxygen for        benzofuran-2-sulfonyl chloride derivatives; and    -   R′, R1, R2, R3, and R4 are as defined above.

For an example of preparing derivatives having formula II,N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-glycine derivatives aresynthesized by selective reaction of primary amine of 2-aminoglycinederivatives prepared by known methods (for instance, where R1 is H, seeS. A. Thomson et al., Tetrahedron, 1995, 6179-6194; where R5 is a sidechain of a protected or unprotected natural or unnatural amino acid, seeA. Puschl et al., Tetrahedron Lett., 1998, 39, 4707-4710).Benzothiazole-2-sulfonyl chloride derivatives are prepared by knownmethods (E. Vedejs, et al., J. Am. Chem. Soc., 1996, 118, 9796-9797.).

The coupling reaction for the preparation of N-[2-(benzothiazole,benzoxazole, benzo[b]thiophene, orbenzofuran-2-sulfonylamino)-ethyl]-glycine derivatives is performed byslow addition of benzothiazole-2-sulfonyl chloride,benzoxazole-2-sulfonyl chloride, benzo[b]thiophene-2-sulfonyl chloride,or benzofuran-2-sulfonyl chloride to a solution ofN-(2-aminoethyl)-glycine derivatives in the presence of non-nucleophilicorganic base at ambient temperature. Examples of solvents of abovereaction include without limitation water, toluene, benzene,ethylacetate, tetrahydrofuran, diisopropylether, diethylether,dichloromethane, chloroform, carbon tetrachloride, and acetonitrile.Preferred solvent is dichloromethane. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylamine,tripropylamine N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base istriethylamine. After completion of the reaction by monitoring by thinlayer chromatography (TLC), the reaction mixture is washed with water,dried, and evaporated in reduced pressure to give the desired product.

Preparation of 1-(benzothiazole, benzoxazole, benzo[b]thiophene orbenzofuran-2-sulfonyl)-piperazin-2-ones

The first precursor synthons having formula IV for synthesis of monomershaving general formula I are prepared from N-[2-(benzothiazole,benzoxazole, benzo[b]thiophene, orbenzofuran-2-sulfonylamino)-ethyl]-glycine derivatives having theformula II by hydrolysis, protection of secondary amine, cyclization,and deprotection of protecting group of secondary amine (FIG. 4).

The entities represented by E, J, R1, R2, R3, R4, R5, and HX may be asdefined above.

First, N-[2-(benzothiazole, benzoxazole, benzo[b]thiophene, orbenzofuran-2-sulfonylamino)-ethyl]-glycine derivatives having theformula II are converted to corresponding acids by adding excesshydroxide ion source. Preferred R6 in the formula II is methyl or ethyl.Examples of hydroxide ion sources include, but are not limited to,lithium hydroxide, sodium hydroxide, and potassium hydroxide. Preferredhydroxide ion source is lithium hydroxide. The reaction mixture withoutwork-up is then treated with a protecting group such as di-t-butyldicarbonate to protect secondary amine to obtain a compound having thegeneral formula:

The entities represented by E, J, R1, R2, R3, R4, and R5 are as definedabove.

Preferred hydrolysis reaction is carried out by adding an aqueoussolution of lithium hydroxide (2 equivalent) to a solution ofN-[2-(benzothiazole, benzoxazole, benzo[b]thiophene, orbenzofuran-2-sulfonylamino)-ethyl]-glycine ester derivative at ambienttemperature. After completion of the reaction by TLC analysis, anaqueous solution of lithium hydroxide (additional 1 equivalent) is addedto the reaction mixture. The reaction mixture is stirred for sufficienttime. Then the excess di-t-butyl dicarbonate is removed by extractionwith ethylacetate. The aqueous solution is then acidified, extractedwith dichloromethane, dried, and evaporated in reduced pressure to yielda solid. Examples of solvents of above reaction are aqueoustetrahydrofuran, aqueous dioxane, and aqueous 1,2-dimethoxyethane.Preferred solvent is aqueous tetrahydrofuran.

Second, the cyclization reaction of carboxylic acids having generalformula III and followed by deprotection of t-Boc produces piperazinonederivatives having general formula IV The cyclization reaction occurssimultaneously during activation of carboxylic acid. The activation ofcarboxylic acid can be conducted by general coupling reagent for peptidesynthesis at ambient temperature. Examples of coupling reagents include,but are not limited to, HATU, HOAt, HODhbt (L. A. Carpino et al., J. Am.Chem. Soc., 1993, 115, 4397-4398), HAPyU, TAPipU (A. Ehrlich et al.,Tetrahedron Lett., 1993, 4781-4784), HBTU (V. Dourtoglou et al.,Synthesis, 1984, 572-574), TBTU, TPTU, TSTU, TNTU (R. Knorr et al.,Tetrahedron Lett., 1989, 1927-1930), TOTU, BOP (B. Castro et al.,Tetrahedron Lett., 1975, 1219-1222), PyBOP (J. Coste et al., TetrahedronLett., 1990, 205-208), BroP (J. Coste et al., Tetrahedron Lett., 1990,669-672), PyBroP (J. Coste et al., Tetrahedron Lett., 1991, 1967-1970),BOI (K. Akaji et al., Tetrahedron Lett., 1992, 3177-3180), MSNT (B.Blankemeyer-Menge et al., Tetrahedron Lett., 1990, 1701-1704), TDO (R.Kirstgen et al., J. Chem. Soc., Chem. Commun., 1987, 1870-1871), DCC,EDCI, CDI (H. A. Staab, Justus Liebigs Ann. Chem., 1957, 609, 75.), HOBt(W König et al., Chem. Ber., 1970, 103, 788, 2024, 2034), HOSu (E.Wünsch et al., Chem. Ber., 1966, 99, 110), NEPIS (R. B. Woodward et al.,J. Am. Chem. Soc., 1961, 83, 1010), BBC (S. Chen et al., TetrahedronLett. 1992, 33, 647), BDMP (P, Li et al., Chem. Lett., 1999, 1163), BOMI(P, Li et al., Tetrahedron Lett., 1999, 40, 3605), AOP (L. A Carpino etal., Tetrahedron Lett., 1994, 35, 2279), BDP (S. Kim et al., TetrahedronLett., 1985, 26, 1341), PyAOP (F. Albericio et al., Tetrahedron Lett.1997, 38, 4853), TDBTU (R. Knorr et al., Tetrahedron Lett. 1989, 30,1927), BEMT (P. Li et al., Tetrahedron Lett. 1999, 40, 8301), BOP-Cl (J.Diago-Meseguer et al., Synthesis 1980, 547), BTFFH, TFFH (A. El-Faham etal. Chem. Lett., 1998, 671), CIP (K. Akaji, et al., Tetrahedron Lett.,1994, 35, 3315), DEPBT (H. Li et al. Organic Lett. 1999, 1, 91), Dpp-Cl(R. Ramage et al., J. Chem. Soc., Perkin Trans 1, 1985, 461), EEDQ (BBelleau et al., J. Am. Chem. Soc., 1968, 90, 1651), FDPP (S Chen et al.,Tetrahedron Lett., 1991, 32, 6711), HOTT, TOTT (M. A. Bailen et al., J.Org. Chem. 1999, 64, 8936), PyCloP (J. Coste et al., Tetrahedron Lett.,1991, 32, 1967 and J. Coste et al., J. Org. Chem., 1994, 59, 2437). Thesolvents may be selected from tetrahydrofuran, dichloromethane,chloroform, DMF, and N-methylpyrrolidone. Preferred solvent is DMF.

Alternatively, the activation of carboxylic acid can be conducted byformation of mixed anhydride using alkyl chloroformate or alkanoylchloride with non-nucleophilic organic base. Examples of alkylhaloformates or alkanoyl chlorides include, but are not limited to,methyl chloroformate, ethyl chloroformate, propyl chloroformate, butylchloroformate, isobutyl chloroformate, pivaloyl chloride, and adamantinecarboxyl chloride. The most preferred acid chloride is isobutylchloroformate. The cyclization reaction using isobutyl chloroformate iscarried out by slowly adding isobutyl chloroformate to a reactionsolution of carboxylic acid having general formula III andnon-nucleophilic organic base in an anhydrous appropriate solvent at thetemperature between −30° C. and 10° C. Examples of non-nucleophilicorganic bases include, but are not limited to, triethylamine,tripropylamine, N,N-diisopropylethylamine, N-methylmorpholine, andN-ethylmorpholine. Preferred non-nucleophilic organic base isN-methylmorpholine. Examples of anhydrous appropriate solvents include,but are not limited to, acetonitrile, chloroform, dichloromethane,1,2-dimethoxy ethane, diethyl ether, diisoproyl ether, andtetrahydrofuran. Preferred solvents are dichloromethane andtetrahydrofuran. The most preferred reaction temperature is in which thereaction mixture is allowed to slowly warm to 0° C. after completingaddition of isobutyl chloroformate at −20° C.

With reference to FIG. 4, the t-Boc group is deprotected in the presenceof acid. Examples of acids include, but are not limited to, HCl, HBr,HF, HI, nitric acid, sulfuric acid, methanesulfonic acid, TFA, andtrifluoromethanesulfonic acid. Preferred acid is TFA. The solvents usedin the deprotection reaction may include without limitationdichloromethane, chloroform, carbon tetrachloride, ethyl acetate,toluene, and benzene. Preferred is dichloromethane.

Synthesis of PNA Monomer

According to a method of this invention, PNA monomers having generalformula I may be synthesized by at least two methods. With reference toFIG. 5, the first approach to PNA monomer syntheses is a method thatintroduces protected or unprotected nucleobase acetic acid moieties toprotected linear backbone prior to cyclization reaction. Alternatively,PNA monomers may be synthesized by beginning with cyclization ofprotected linear backbone, followed by coupling of protected orunprotected nucleobase acetic acid moieties to create desired products.

Method 1

The linear moieties having general formula V are prepared from protectedlinear backbone having general formula II by acylation of nucleobaseacetic acid moieties using coupling reagents as shown in FIG. 5.

With reference to FIG. 5, the coupling reaction was conducted byaddition of coupling reagent to the mixture of protected linear backbonehaving general formula II, nucleobase acetic acid moieties, andnon-nucleophilic organic base in anhydrous appropriate solvent. Examplesof coupling reagents include, but are not limited to, HATU, HOAt,HODhbt, HAPyU, TAPipU, HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP, PyBOP,BroP, PyBroP, BOI, MSNT, TDO, DCC, EDCI, CDI, HOBt, HOSu, NEPIS, BBC,BDMP, BOMI, AOP, BDP, PyAOP, TDBTU, BOP-Cl, CIP, DEPBT, Dpp-Cl, EEDQ,FDPP, HOTT, TOTT, PyCloP. Preferred coupling reagent is PyBOP. Examplesof non-nucleophilic organic bases include, but are not limited to,triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N,N-diisopropylethylamine. Examples of anhydrousappropriate solvents include, but are not limited to, chloroform,dichloromethane, 1,2-dimethoxyethane, tetrahydrofuran, DMF, andN-methylpyrrolidone. Preferred solvent is DMF.

The entities represented by E, J, R1, R2, R3, R4, R5, R6, and B are asdefined above.

Compounds having the general formula V are converted to correspondingacids such as formula VI by adding an excess of hydroxide ion source.Preferably, R6 may be a methyl, ethyl or t-butyl. Examples of hydroxideion sources include, but are not limited to, lithium hydroxide, sodiumhydroxide, and potassium hydroxide. Preferred hydroxide ion source islithium hydroxide.

The entities represented by E, J, R1, R2, R3, R4, R5, and B are asdefined above.

Further, with reference to FIG. 5, the cyclization reaction ofcarboxylic acids produces PNA monomers having the general formula I bysimultaneous reaction during activation of, carboxylic acid. Theactivation of carboxylic acid may be conducted by general couplingreagent for peptide synthesis at ambient temperature. Examples ofcoupling reagents include, but are not limited to, HATU, HOAt, HODhbt,HAPyU, TAPipU, HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP, PyBOP, BroP,PyBroP, BOI, MSNT, TDO, DCC, EDCI, CDI, HOBt, HOSu, NEPIS, BBC, BDMP,BOMI, AOP, BDP, PyAOP, TDBTU, BOP-Cl, CIP, DEPBT, Dpp-Cl, EEDQ, FDPP,HOTT, TOTT, PyCloP. Preferred coupling reagent is PyBOP. Examples ofnon-nucleophilic organic bases include, but are not limited to,triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N,N-diisopropylethylamine. The solvents may be withoutlimitation selected from tetrahydrofuran, dichloromethane, chloroform,DMF, or N-methylpyrrolidone. Preferred solvent is DMF.

Alternatively, the activation of carboxylic acid can be conducted byformation of mixed anhydride using alkyl chloroformate or alkanoylchloride with non-nucleophilic organic base. Examples of alkylhaloformates or alkanoyl chlorides include, but are not limited to,methyl chloroformate, ethyl chloroformate, propyl chloroformate, butylchloroformate, isobutyl chloroformate, pivaloyl chloride, and adamantinecarboxyl chloride. The most preferred acid chloride is isobutylchloroformate. The cyclization reaction using isobutyl chloroformate iscarried out by slowly adding isobutyl chloroformate to a reactionsolution of carboxylic acid and non-nucleophilic organic base in ananhydrous appropriate solvent at a temperature between −30° C. and 10°C. Examples of non-nucleophilic organic bases include, but are notlimited to, triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N-methylmorpholine. Examples of anhydrous appropriatesolvents include, but are not limited to, acetonitrile, chloroform,dichloromethane, 1,2-dimethoxyethane, diethyl ether, diisoproyl ether,and tetrahydrofuran. Preferred solvents are dichloromethane andtetrahydrofuran. Preferred reaction temperature is in which the reactionmixture is allowed to slowly warm to about 0° C. after completingaddition of isobutyl chloroformate at −20° C.

Method 2

As an alternative method, PNA monomers according to this invention maybe prepared by coupling of protected or unprotected nucleobase aceticacid moieties to cyclic precursor having the general formula IV:

The entities represented by E, J, R1, R2, R3, R4, and R5 are as definedabove.

With reference to FIG. 6, the coupling reaction of cyclic precursor withprotected or unprotected nucleobase acetic acid moieties is carried outby using general coupling reagent for peptide synthesis andnon-nucleophilic organic bases at ambient temperature. Examples ofcoupling reagents include, but are not limited to, HATU, HOAt, HODhbt,HAPyU, TAPipU, HBTU, TBTU, TPTU, TSTU, TNTU, TOTU, BOP, PyBOP, BroP,PyBroP, BOI, MSNT, TDO, DCC, EDCI, CDI, HOBt, HOSu, NEPIS, BBC, BDMP,BOMI, AOP, BDP, PyAOP, TDBTU, BOP-Cl, CIP; DEPBT, Dpp-Cl, EEDQ, FDPP,HOTT, TOTT, PyCloP. Preferred coupling reagent is PyBOP. Examples ofnon-nucleophilic organic bases include, but are not limited to,triethylamine, tripropylamine, N,N-diisopropylethylamine,N-methylmorpholine, and N-ethylmorpholine. Preferred non-nucleophilicorganic base is N,N-diisopropylethylamine. The solvent may be withoutlimitation tetrahydrofuran, dichloromethane, chloroform, DMF, orN-methylpyrrolidone. Preferred solvent is DMF.

Nucleobases and Protecting Group

Examples of nucleobases of this invention include, but are not limitedto, adenine, cytosine, guanine, thymine, uridine, 2,6-diaminopurine, andnaturally or non-naturally occurring nucleobases as depicted in FIG. 1and FIG. 2. Preferred nucleobases are adenine, cytosine, guanine, andthymine. Nucleobases may be protected by protecting group for thesyntheses of PNA oligomers. Protecting groups may be, but are notlimited to, Boc, adamantyloxycarbonyl, benzyloxycarbonyl (P. E. Nielsenet al., Science, 1991, 254, 1497-1500; M. Egholm et al., J. Am. Chem.Soc., 1992, 114, 9677-9679; M. Egholm et al., J. Am. Chem. Soc., 1992,114, 1895-1897, M. Egholm et al., J. Chem. Soc. Chem. Commun., 1993,800-801; K. L. Dueholm et al., J. Org. Chem., 1994, 59, 5767-5773; WO92/20702), 4-methoxybenzyloxycarbonyl, 3,4-dimethoyxbenzyloxycarbonyl,benzhydryloxycarbonyl (U.S. Pat. No. 6,133,444), piperonyloxycarbonylderivatives, 2-methylthioethoxycarbonyl (U.S. Pat. No. 6,063,569); Mmt(G. Breipohl et al., Bioorg. Med. Chem. Lett., 1996, 6, 665-670), oracid labile protecting group (T. W. Greene and P. G. M. Wuts, ProtectiveGroup in Organic Synthesis, 3^(rd) Edition, pp 494-653).

Synthesis of T-Monomer

T-monomer is a compound having general formula I-t:

The entities represented by E, J, R1, R2, R3, R4, and R5 are as definedabove. Preferably E is nitrogen, and J is sulfur.

The precursor for T-monomer, (thymin-1-yl)-acetic acid (shown below), isprepared by known methods (K. L. Dueholm et. al., J. Org. Chem., 1994,59, 5767-5773; WO 92/20702).

With reference to FIG. 8, the compounds of general formula V-t areprepared by coupling reaction of (thymin-1-yl)-acetic acid withbenzothiazole-2-sulfonyl, benzoxazole-2-sulfonyl,benzo[b]thiophene-2-sulfonyl or benzofuran-2-sulfonyl group protectedbackbone ester having general formula II in the presence of couplingreagent to afford the compound having general formula:

The entities represented by E, J, R1, R2, R3, R4, R5, and R6 are asdefined above. Preferably E is nitrogen, and J is sulfur.

Preferred R6 is methyl, ethyl or t-butyl.

The compounds having the general formula V-t are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-t.

The entities represented by E, J, R1, R2, R3, R4, and R5 are as definedabove. Preferably E is nitrogen, and J is sulfur.

With reference to FIG. 8, the cyclization reaction of carboxylic acidsproduces PNA T-monomers having general formula I-t by simultaneousreaction during activation of carboxylic acid. The activation ofcarboxylic acid may be conducted by general coupling reagent for peptidesynthesis or mixed anhydride. The reaction conditions and reagents arethe same as described above.

Alternatively, as seen in FIG. 9, PNA T-monomers can be prepared bycoupling (thymin-1-yl)-acetic acid to piperazinone derivatives havinggeneral formula IV. The reaction conditions and reagents are the same asdescribed above.

Synthesis of C-Monomer

C-monomer is a compound having general formula I-c:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The precursors for PNA C-monomers, suitably protected(cytosin-1-yl)-acetic acids (shown below), are prepared by the methodaccording to the scheme as depicted in FIG. 7 and known methods such asdescribed in U.S. Pat. No. 6,133,444; U.S. Pat. No. 6,063,569; Dueholm,et al., J. Org. Chem., 1994, 59, 5767-5773; and WO 92/20702, which areincorporated by reference herein in their entirety, or modificationsthereof.

-   -   R7 may be methyl, ethyl, benzyl, benzhydryl, 4-methoxybenzyl,        3,4-dimethoxybenzyl, piperonyl derivatives, or 2-methylthioethyl        group.

With reference to FIG. 10, PNA C-monomer is prepared by couplingreaction of suitably protected (cytosin-1-yl)-acetic acids with abenzothiazole-2-sulfonyl, benzoxazole-2-sulfonyl,benzo[b]thiophene-2-sulfonyl or benzofuran-2-sulfonyl group protectedbackbone ester having general formula II in the presence of couplingreagent to afford the compound having general formula:

The entities represented by E, J, R1, R2, R3, R4, R5, R6, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The compounds having the general formula V-c are converted tocorresponding acids by adding an excess of hydroxide ion source toobtain a compound having general formula VI-c:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

With reference to FIG. 10, the cyclization reaction of carboxylic acidsproduces PNA monomers having the general formula I-c by simultaneousreaction during activation of carboxylic acid. The activation ofcarboxylic acid may be conducted by general coupling reagent for peptidesynthesis or mixed anhydride. The reaction conditions and reagents arethe same as described above.

Alternatively, as seen in FIG. 11, PNA C-monomer can be prepared bycoupling suitably protected (cytosin-1-yl)-acetic acids to piperazinonederivatives having general formula IV. The reaction conditions andreagents are the same as described above.

Synthesis of A-Monomer

A-monomer is a compound having general formula I-a:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The precursors for PNA A-monomers, suitably protected(adenin-9-yl)-acetic acids (shown below), are prepared by the methodaccording to the scheme as depicted in FIG. 7 and known methods such asdescribed in U.S. Pat. No. 6,133,444; and S. A. Thomson et al.,Tetrahedron, 1995, 6179-6194, which are incorporated by reference hereinin their entirety, or modifications thereof.

-   -   R7 is selected from methyl, ethyl, benzyl, benzhydryl,        4-methoxybenzyl, 3,4-dimethoxybenzyl, piperonyl derivatives, and        2-methylthioethyl group.

With reference to FIG. 12, PNA C-monomer is prepared by couplingreaction of suitably protected (adenin-9-yl)-acetic acids with abenzothiazole-2-sulfonyl, benzoxazole-2-sulfonyl,benzo[b]thiophene-2-sulfonyl or benzofuran-2-sulfonyl group protectedbackbone ester having general formula II in the presence of couplingreagent to obtain the compound having general formula V-a:

The entities represented by E, J, R1, R2, R3, R4, R5, R6, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The compounds having the general formula V-a are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-a:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

With reference to FIG. 12, the cyclization reaction of carboxylic acidsproduces PNA monomers having the general formula I-a by simultaneousreaction during activation of carboxylic acid. The activation ofcarboxylic acid can be conducted by general coupling reagent for peptidesynthesis or mixed anhydride. The reaction conditions and reagents arethe same as described above.

Alternatively, as seen in FIG. 13, PNA A-monomer can be prepared bycoupling suitably protected (adenin-9-yl)-acetic acids to piperazinonederivatives having general formula IV. The reaction conditions andreagents are the same as described above.

Synthesis of G-monomer

G-monomer is a compound having general formula I-g:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The precursors for PNA G-monomers, suitably protected(guanin-9-yl)-acetic acids (shown below), are prepared by the methodaccording to the scheme depicted in FIG. 7 and known methods such asdescribed in U.S. Pat. No. 6,172,226, or modifications thereof.

-   -   R7 may be methyl, ethyl, benzyl, benzhydryl, 4-methoxybenzyl,        3,4-dimethoxybenzyl, piperonyl derivatives, or 2-methylthioethyl        group.

With reference to FIG. 14, PNA G-monomer is prepared by couplingreaction of suitably protected (guanin-9-yl)-acetic acids with abenzothiazole-2-sulfonyl, benzoxazole-2-sulfonyl,benzo[b]thiophene-2-sulfonyl or benzofuran-2-sulfonyl group protectedbackbone ester having general formula II in the presence of couplingreagent to afford the compound having general formula V-g:

The entities represented by E, J, R1, R2, R3, R4, R5, R6, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

The compounds having the general formula V-g are converted tocorresponding acids by adding an excess hydroxide ion source to affordthe compound having general formula VI-g:

The entities represented by E, J, R1, R2, R3, R4, R5, and R7 are asdefined above. Preferably E is nitrogen, and J is sulfur.

With reference to FIG. 14, the cyclization reaction of carboxylic acidsproduces PNA monomers having the general formula I-g by simultaneousreaction during activation of carboxylic acid. The activation ofcarboxylic acid can be conducted by general coupling reagent for peptidesynthesis or mixed anhydride. The reaction conditions and reagents arethe same as described above.

Alternatively, as seen in FIG. 15, PNA G-monomer can be prepared bycoupling suitably protected (guanin-9-yl)-acetic acids to piperazinonederivatives having general formula IV. The reaction conditions andreagents are the same as described above.

Synthesis of PNA Oligomers

Various combinatorial synthetic methods already reported in chemicalliterature are generally applicable to PNA oligomer synthesis using themonomers of this invention. These methods include, but are not limitedto, solid phase synthesis and solution phase synthesis. After the PNAmonomers have been synthesized in the manner described above, PNAoligomers are constructed by solid phase synthesis on a suitable supportmaterial such as, but not limited to, polystyrene,polyoxyethylene-modified polystyrene, such as, for example Tentagel® orControlled Pore Glass, which is provided with anchoring group whichlatently contains cleavable amine functional group. In solid phasesynthesis, the first PNA monomer of this invention is incorporated bycoupling reaction to solid support. The next step is systematicelaboration of desired PNA oligomer sequence. This elaboration includesrepeated deprotection/coupling/capping cycles. The backbone protectinggroup on the last coupled monomer, benzothiazole-2-sulfonyl,benzoxazole-2-sulfonyl, benzo[b]thiophene-2-sulfonyl orbenzofuran-2-sulfonyl group, is quantitatively removed by treatment withsuitable thiol in the presence of organic base to liberate terminal freeamine. Once the synthesis of PNA oligomer has been completed, theoligomers are cleaved from the solid support and nucleobase protectinggroups are simultaneously removed by incubation for 1-2 h. at about roomtemperature in TFA containing cresol as a cation scavenger.

Following is an example of a general reaction cycle that may be used forthe synthesis of PNA oligomers, and is not meant to limit the inventionin any way, such as in the sequence of steps, since any oligomersynthesis method may be generally used so long as the inventive PNAmonomer is employed.

-   -   1. Removing protecting group from resin to activate amine        functional group.    -   2. Incorporating amino-acid, linker, or PNA monomer having        terminal protected amine group to resin.    -   3. Washing.    -   4. Capping with acetic anhydride in the presence of organic        base.    -   5. Washing.    -   6. Cleavage over reacted acetyl group in sulfonamide.    -   7. Washing.    -   8. Deprotecting sulfonyl group.    -   9. Washing.    -   10. Adding monomer.    -   11. Returning to No. 3 and repeating No. 4-No. 11.

In the course of the coupling reaction of monomer for the oligomersynthesis reaction, the acylating reaction can be accelerated by using acatalyst such as but not limited to mercury acetate, tetramethylammoniumfluoride, tetraethylammonium fluoride, tetrabutylammonium fluoride,benzyltrimethylammonium fluoride, cesium fluoride, tributylphosphine,triphenylphosphine. Preferred catalyst is tetrabutylammonium fluoride.Also, the reaction rate depends on the solvent used and reactiontemperature. Examples of solvents include, but are not limited to, DMF,N-methylpyrrolidone, dimethoxyethane, dichloromethane,1,2-dichloroethane, DMSO, tetrahydrofuran, hexamethylphophoramide,tetramethylene sulfone, isopropyl alcohol, ethyl alcohol, and mixture ofselected sovents. Preferred solvent is DMF. The N-terminal aminoprotecting group is cleaved by using thiol with organic base in solvent.Examples of thiols include, but are not limited to, C₂˜C₂₀ alkanethiol,4-methoxytoluenethiol, 4-methylbenzenethiol, 3,6-dioxa-1,8-octanethiol,4-chlorotoluenethiol, benzylmercaptane, N-acetylcysteine,N-(t-Boc)cysteine methyl ester, methyl 3-mercaptopropionate,4-methoxybenzene thiol. Examples of organic bases include, but are notlimited to, triethylamine, N,N-diisopropyethylamine, piperidine,N-methylmorpholine, and 1,8-diazabicyclo[5,4,0]undec-7-one. Preferredorganic base is N,N-diisopropyethylamine.

LIST OF ABBREVIATIONS

-   -   t-Boc tert-Butyloxycarbonyl    -   AOP O-(7-azabenzotriazol-1-yl)-tris(dimethylamino)phosphonium    -   BBC 1-benzotriazol-1-yloxy-bis(pyrrolidino)uronium        hexafluorophosphate    -   BDMP 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl        2H-pyrrolium hexachloroanitimonate    -   BDP benzotriazol-1-yl diethyl phosphate    -   BEMT 2-bromo-3-ethyl-4-methyl thiazolium tetrafluoroborate    -   BTFFH bis(tetramethylenefluoroformamidinium)hexafluorophosphate    -   BOMI benzotriazol-1-yloxy-N,N-dimethylmethaniminium        hexachloroantimonate    -   BOI 2-(benzotriazol-1-yl)oxy-1,3-dimethyl-imidazolinium        hexafluorophosphate    -   BOP benzotriazolyl-1-oxy-tris(dimethylamino)phophonium        hexafluorophosphate    -   BOP-Cl bis(2-oxo-3-oxazolidinyl)phosphinic chloride    -   BroP bromotris(dimethylamino)phophonium hexafluorophosphate    -   CDI carbonyldiimidazole    -   CIP 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate    -   DMF dimethylformamide    -   DCC 1,3-dicyclohexylcarbodiimide    -   DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4 (3H)-one    -   Dpp-Cl diphenylphosphinic chloride    -   EDCI 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide        hydrochloride    -   EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline    -   Fmoc 9-fluorenylmethyloxycarbonyl    -   FDPP pentafluorophenyl diphenylphosphinate    -   HAPyU        O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uranium        hexafluorophosphate    -   HATU O-(7-azabenzotriazol-1-yl)-1,3,3-tetramethyluranium        hexafluorophosphate    -   HBTU O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluranium        hexafluorophosphate    -   HOBt hydroxybenzotriazole    -   HOAt 1-hydroxy-7-azabenzotriazole    -   HODhbt 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine    -   HOSu hydroxysuccinimide    -   HOTT S-(1-oxido-2-pyridinyl)-1,1,3,3-tetramethylthiouronium        hexafluorophosphate    -   MSNT 2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide    -   Mmnt 47-methoxyphenyldiphenylmethyl    -   NEPIS N-ethyl-5-phenylisoxazolium-3′-sulfonate    -   PyAOP 7-azobenzotriazolyoxytris(pyrrolidino)phosphonium        hexafluorophosphate    -   PyBOP benzotriazolyl-1-oxy-tripyrrolidinophosphonium        hexafluorophosphate    -   PyBroP bromotripyrrolidinophosphonium hexafluorophosphate    -   PyCloP chlorotris(pyrrolydino)phophonium hexafluorophosphate    -   TAPipU        O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uranium        tetrafluoroborate    -   TBTU O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluranium        tetrafluoroborate    -   TDO 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide    -   TFA trifluoroacetic acid    -   TFFH tetramethylfluoroformamidinium hexafluorophosphate    -   TOTT S-(1-oxido-2-pyridinyl)-1,1,3,3-tetramethylthiouronium        tetrafluoroborate    -   TDBTU        2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium    -   tetrafluoroborate    -   TNTU        O-[(5-norbonene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TOTU        O-[(cyano(ethoxycarbonyl)methylene)amino]-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TPTU O-(1,2-dihydro-2-oxo-1-pyridyl-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TSTU O-(N-succinimidyl)-1,1,3,3-tetramethyluronium        tetrafluoroborate

This invention is more specifically illustrated by following Examples,which are not meant limit the invention, unless otherwise noted.

EXAMPLES Example 1 N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester

To a solution of N-(2-aminoethyl)-glycine ethyl ester 2HCl (1.10 g, 5.0mmol), prepared as described by Will (D. W. Will et al., Tetrahedron,1995, 51, 12069.), in dichloromethane (50 mL) was slowly addedtriethylamine (2.02 g, 20 mmol) at room temperature. Thenbenzothiazole-2-sulfonyl chloride (1.19 g 5.0 mmol) in dichloromethane(10 mL) was added to the reaction mixture at room temperature for 5 min.The resulting reaction mixture was stirred for additional 2 h, at roomtemperature and washed with water (30 mL). The organic layer was driedover MgSO₄ and filtered. The filtrate was evaporated to remove solventto give desired product (1.60 g, 92%) as a solid. ¹H NMR (500 MHz;DMSO-d₆) δ 8.28 (d, 1H), 8.19 (d, 1H), 7.69-7.63 (m, 2H), 4.03 (q, 2H),3.24 (s, 2H), 3.13 (t, 2H), 2.62 (t, 2H), 1.15 (t, 3H).

Example 2 [2-(5-Chloro-benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester

The title compound (657 mg, 87%) was synthesized by the reaction ofN-(2-aminoethyl)-glycine ethyl ester 2HCl (398 mg, 2.0 mmol) with5-chloro-benzothiazole-2-sulfonyl chloride (536 mg, 2.0 mmol) as per theprocedure of example 1. ¹H NMR (500 MHz; DMSO-d₆) δ 8.32 (d, 1H), 8.31(s, 1H), 7.71 (d, 1H), 4.02 (q, 2H), 3.25 (s, 2H), 3.14 (t, 2H), 2.62(t, 2H), 1.15 (t, 3H).

Example 3 [2-(4-Chloro-5-methoxybenzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester

The title compound (726 mg, 89%) was synthesized by the reaction ofN-(2-aminoethyl)-glycine ethyl ester 2HCl (398 mg, 2.0 mmol) with4-chloro-5-methoxy-benzothiazole-2-sulfonyl chloride (596 mg, 2.0 mmol)as per the procedure of Example 1. ¹H NMR (500 MHz; DMSO-d₆) δ 8.18 (d,1H), 7.57 (d, 1H), 4.00 (q, 2H), 3.97 (s, 3H), 3.22 (s, 2H), 3.14 (t,2H), 2.62 (t, 2H), 1.13 (t, 3H).

Example 4 N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-alanine ethylester

N-(2-Aminoethyl)-alanine ethylester 2HCl, prepared as described byPuschl (A. Puschl et al., Tetrahedron, 1998, 39, 4707.), was reactedwith benzothiazole-2-sulfonyl chloride as per the procedure of Example 1to give the title compound. ¹H NMR (500 MHz DMSO-d₆) δ 8.27 (d, 1H),8.18 (d, 1H), 7.69-7.62 (m, 2H), 4.01 (q, 2H), 3.17 (q, 1H), 3.13 (t,2H), 2.61 (m, 1H), 2.49 (m, 1H), 1.13 (t, 3H), 1.06 (d, 3H).

Example 5N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine

To a solution of N-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester (6.87 g, 20 mmol) in tetrahydrofuran (50 mL) was added asolution of LiOH (1.64 g, 40 mmol) dissolved in water (30 mL). Afterstirring for 1 hour at room temperature, di-t-butyl dicarbonate (6.55 g,30 mmol) was added to the reaction mixture. The resulting reactionmixture was stirred for 30 min. and then a solution of LiOH (0.82 g,0.02 mol) in water (15 mL) was added. After completion of the reactionby TLC, the precipitate was removed by filtration and tetrahydrofuranwas removed in vacuo. The residual solution was washed with ethyl ether(100 mL). The aqueous layer was acidified to pH 3 by adding 2N HCl andextracted with dichloromethane (100 mL). The organic layer was driedover MgSO₄ and filtered. The filtrate was concentrated in vacuo toafford the desired product (7.88 g, 95%). ¹H NMR (500 MHz; DMSO-d₆) δ8.73 (d, 1H), 8.19 (dd, 1H), 7.71-7.63 (m, 2H), 3.84 (s, 1H), 3.79 (s,1H), 3.30-3.22 (m, 4H), 1.34 (s, 4.5H), 1.28 (s, 4.5H).

Example 6N-[2-(5-Chloro-benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine

The title compound (607 mg, 90%) was synthesized fromN-[2-(5-chloro-benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester(567 g, 1.5 mmol) as per the procedure of Example 5. ¹H NMR (500 MHz;DMSO-d₆) δ 8.80 (br, 1H), 8.33 (d, 1H), 8.32 (s, 1H), 7.72 (d, 1H), 3.84(s, 1H), 3.80 (s, 1H), 3.29 (m, 2H), 3.23 (m, 2H), 1.35 (s, 4.5H), 1.28(s, 4.5H).

Example 7N-[2-(4-Chloro-5-methoxy-benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine

The title compound (655 mg, 91%) was synthesized fromN-[2-(4-chloro-5-methoxy-benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester (612 g, 1.5 mmol) as per the procedure of Example 5. ¹H NMR(500 MHz; DMSO-d₆) δ 8.83 (br, 1H), 8.20 (d, 1H), 7.58 (d, 1H), 3.98 (s,3H), 3.83 (s, 1H), 3.79 (s, 1H), 3.32-3.23 (m, 4H), 1.34 (s, 4.5H), 1.25(s, 4.5H).

Example 8N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-alanine

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-alanine ethyl ester as perthe procedure of Example 5. ¹H NMR (500 MHz; DMSO-d₆) δ 8.85 (brs, 1H),8.28 (d, 1H), 8.18 (dd, 1H), 7.71-7.64 (m, 2H), 4.33 (q, 0.5H), 4.07 (q,1H), 3.34-3.15 (m, 4H), 1.34 (s, 4.5H), 1.32 (s, 4.5H), 1.29 (d, 2H).

Example 9 1-(Benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroaceticacid salt

To a solution ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine(8.30 g, 20 mmol) in dichloromethane (100 mL) was added dicyclohexylcarbodiimide (5.16 g, 25 mmol). After stirring for 2 hours at roomtemperature, the precipitate was removed by filtration. The filtrate wasconcentrated to approximately 30 mL and cooled to 0° C. To a coldsolution was added trifluoroacetic acid (20 mL). The mixture was stirredfor 2 h at the same temperature and ethyl ether (100 mL) was slowlyadded. The precipitate product was filtered off, washed with ethylether, and dried in vacuo to afford white solid (6.58 g, 80%). ¹H-NMR(500 MHz DMSO-d₆) δ 8.36 (m, 1H), 8.25 (m, 1H), 7.76-7.70 (m, 2H), 4.17(t, 2H), 3.96 (s, 2H), 3.56 (t, 2H).

Example 10 1-(5-Chloro-benzothiazole-2-sulfonyl)-piperazin-2-onetrifluoroacetic acid salt

The title compound (342 mg, 78%) was synthesized fromN-[2-(5-chloro-benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine(540 mg, 1.2 mmol) as per the procedure of Example 9. ¹H-NMR (500 MHz;DMSO-d₆) δ 9.55 (br, 2H), 8.40 (d, 1H), 8.39 (d, 1H), 7.79 (dd, 1H),4.16 (t, 2H), 3.97 (s, 2H), 3.57 (t, 2H).

Example 111-(4-Chloro-5-methoxy-benzothiazole-2-sulfonyl)-piperazin-2-onetrifluoroacetic acid salt

The title compound (389 mg, 82%) was synthesized fromN-[2-(4-chloro-5-methoxy-benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-glycine(576 mg, 1.2 mmol) as per the procedure of Example 9. ¹H-NMR (500 MHz;DMSO-d₆) δ 8.28 (d, 1H), 7.68 (d, 1H), 4.16 (dd, 2H), 3.99 (s, 3H), 3.96(s, 2H), 3.56 (dd, 2H).

Example 12 1-(Benzothiazole-2-sulfonyl)-3-methyl-piperazin-2-onetrifluoroacetic acid salt

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-(tert-butoxycarbonyl)-alanineas per the procedure of Example 9. ¹H-NMR (500 MHz; DMSO-d₆) δ 9.80(brs, 2H), 8.35 (m, 1H), 8.25 (m, 1H), 7.75-7.70 (m, 2H), 4.31-4.25 (m,2H), 4.17 (m, 1H), 3.70 (m, 1H), 3.55 (m, 1H), 1.34 (d, 3H).

Example 13 [4-N-(Piperonyloxycarbonyl)-cytosin-1-yl]-acetic acid ethylester

The reaction mixture of (cytosin-1-yl)-acetic acid (3.94 g, 20 mmol) and1,1′-carbonyldiimidazole (4.86 g, 30 mmol) in DMF was stirred for 25min. at 100° C. Then, piperonyl alcohol (6.08 g, 40 mmol) was added tothe reaction mixture. The resulting reaction mixture was allowed to coolto room temperature for 2 h. The solvent was removed in vacuo and theresidue was dissolved in ethyl acetate (100 mL). The solution was washedwith water (100 mL×2), dried over MgSO₄, and filtered. The filtrate wasconcentrated in vacuo and treated with ethyl ether to afford whitesolid. The solid was filtered off and dried in vacuo to give the desiredproduct (7.20 g, 96%). ¹H-NMR (500 MHz; DMSO-d₆) δ 10.77 (s, 1H), 8.04(d, 1H), 7.04 (d, 1H), 7.00 (s, 1H), 6.92 (s, 2H), 6.02 (s, 2H), 5.09(s, 2H), 4.61 (s, 2H), 4.15 (q, 2H), 1.19 (t, 3H).

Example 14 [6-N-(Piperonyloxycarbonyl)-adenin-9-yl]-acetic acid ethylester

The title compound (6.8 g, 85%) was synthesized from(adenin-9-yl)-acetic acid ethyl ester (4.42 g, 20 mmol) as per theprocedure of Example 13. ¹H-NMR (500 MHz; DMSO-d₆) δ 10.65 (s, 1H), 8.62(s, 1H), 8.44 (s, 1H), 7.05 (s, 1H), 6.95 (d, 1H), 6.92 (d, 1H), 6.03(s, 2H), 5.20 (s, 2H), 5.12 (s, 2H), 4.18 (q, 2H), 1.22 (t, 3H).

Example 15 (2-Amino-6-iodopurin-9-yl)-acetic acid ethyl ester

To a solution of 2-amino-6-iodo-purine (78.3 g, 0.3 mol) in DMF (1960mL) was added ethyl bromoacetate (55.1 g, 0.33 mol) and potassiumcarbonate (82.9 g, 0.6 mol). The resulting reaction mixture was stirredfor 12 h at room temperature. The reaction mixture was concentrated tosmall volume (about 150 mL) in vacuo and the residue was dissolved inwater. The solid was filtered off, washed with water and ethyl ether,and dried in vacuo to give the titled compound (98.4 g, 95%). ¹H-NMR(500 MHz; DMSO-d₆) δ 8.06 (s, 1H), 6.90 (br.s, 2H), 4.94 (s, 2H), 4.17(q, 2H), 1.22 (t, 3H).

Example 16 [2-(Piperonyloxycarbonyl)-amino-6-iodopurine-9-yl]-aceticacid ethyl ester

To a solution of (2-amino-6-iodopurine-9-yl)-acetic acid ethyl ester(3.47 g, 10 mmol) in tetrahydrofuran (40 mL) was added triphosgene (1.20g, 4 mmol) at 0° C. After stirring for additional 30 min,N,N-diisopropylethylamine (3.30 g) was slowly added and the reactionmixture was stirred for 30 min at 0° C. Then, piperonyl alcohol (2.30 g,15 mmol) was added and the resulting reaction mixture was allowed towarm to room temperature and stirred for additional 1.5 h. To theresulting mixture was added water (50 mL) and ethyl alcohol (50 mL). Thesolution was concentrated to about 100 mL to precipitate solid. Thesolid was filtered off, washed with ethyl alcohol, and dried in vacuo togive the desired product (2.40 g, 46%). ¹H NMR (500 MHz; DMSO-d₆) δ10.77 (s, 0.5H), 10.69 (s, 0.5H), 8.49 (s, 0.5H), 8.45 (s, 0.5H), 7.01(s, 1H), 6.94 (d, 1H), 6.91 (d, 1H), 6.02 (s, 2H), 5.11 (s, 1H), 5.07(s, 2H), 5.06 (s, 1H).

Example 17 [4-N-(Piperonyloxycarbonyl)-cytosin-1-yl]-acetic acid

To a suspension of [4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetic acidethyl ester (6.40 g, 17 mmol) in tetrahydrofuran (30 mL) and water (60mL) was added lithium hydroxide monohydrate (1.6 g) at room temperature.After stirring 20 min, the reaction mixture was acidified by adding 1 NHCl (40 mL). The precipitated solid was filtered off, washed with water(20 mL) and ethyl alcohol (20 mL), and dried in vacuo to give thedesired product (5.8 g, 98%). NH-NMR (500 MHz; DMSO-d₆) δ 7.94 (d, 1H),6.93 (d, 1H), 6.90 (s, 1H), 6.82 (s, 2H), 5.93 (s, 2H), 4.99 (s, 2H),4.43 (s, 2H).

Example 18 [6-N-(Piperonyloxycarbonyl)-adenin-9-yl]-acetic acid

The title compound (3.67 g, 99%) was synthesized from[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetic acid ethyl ester (4.00g, 10 mmol) as per the procedure of Example 17. ¹H NMR MHz; DMSO-d₆) δ10.53 (s, 1H), 8.52 (s, 1H), 8.33 (s, 1H), 6.96 (s, 1H), 6.86 (d, 1H),6.82 (d, 1H), 5.93 (s, 2H), 5.02 (s, 2H), 4.99 (s, 2H).

Example 19 [2-N-(Piperonyloxycarbonyl)-guanin-9-yl]-acetic acid

To a suspension of 60% NaH (1.3 g, 32.5 mmol) in tetrahydrofuran (40 mL)was slowly added 3-hydroxypropionitrile (2.3 g, 29.5 mmol) for a periodof 20 min. at 0° C. and the mixture was stirred for additional 30 min.To the resulting reaction mixture was slowly added[2-(piperonyloxycarbonyl)-amino-6-iodo-purine-9-yl]-acetic acid ethylester (2.4 g, 4.6 mmol) portion-wise in an ice bath. After the additionwas completed, the ice bath was removed and stirring continued foradditional 3 h. Then, water (20 mL) was added and stirred for additional30 min. The reaction mixture was acidified to pH 3 by addition of 1 NHCl solution. After removal of tetrahydrofuran in vacuo, the solutionwas cooled in ice bath and the solid collected by filtration. The solidwas washed with water (20 mL) and ethyl alcohol (20 mL) and dried invacuo to give the desired product (1.4 g, 84%). ¹H NMR (500 MHz;DMSO-d₆) δ 7.73 (s, 1H), 6.91 (s, 1H), 6.84 (d, 1H), 6.82 (d, 1H), 5.93(s, 2H), 5.04 (s, 2H), 4.39 (s, 2H).

Example 20N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester

To the mixture of N-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester (1.72 g, 5 mmol), (thymin-1-yl)-acetic acid (0.92 g, 5mmol), HOBt (0.81 g, 6 mmol), and DCC (1.24 g, 6 mmol) in DMF (15 mL)was added N,N-diisopropylethylamine (1.31 mL, 7.5 mmol) at ambienttemperature. The resulting reaction mixture was stirred for 5 h at thesame temperature and the solvent was removed in vacuo to 5 mL. Theresidue was dissolved in dichloromethane (50 mL) and the precipitate wasfiltered. The filtrate was washed with 1N HCl aqueous solution,saturated sodium bicarbonate solution, and brine. The organic layer wasdried over magnesium sulfate and filtered. The filtrate was concentratedand the residue was triturated with ethyl alcohol. The resulting solidwas filtered off and dried in vacuo to give the title compound (1.91 g,75%) as a white solid. ¹H NMR (500 MHz; DMSO-d₆) δ 11.29 (s, 0.6H),11.28 (s, 0.4H), 8.99 (brs, 0.6H), 8.82 (brs, 0.4H), 8.28 (m, 1H) 8.18(d, 1H) 7.66 (m, 2H) 7.31 (s, 0.6H) 7.42 (s, 0.4H) 4.66 (s, 1.2H) 4.47(s, 0.8H) 4.31 (s, 0.8H), 4.05 (s, 1.2H), 4.04 (q, 1.2H), 3.55 (t,1.2H), 3.40-3.34 (m, 2.8H), 3.20 (t, 0.8H), 1.73 (s, 3H), 1.19 (t,1.2H), 1.14 (t, 1.8H).

Example 21N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester

The title compound (2.99 g, 85%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g;5 mmol) and [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid (1.90g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 11.00 (s, 1H), 8.86 (brs, 114), 8.27 (d, 1H), 8.18 (d, 1H), 7.89 (d,0.6H), 7.83 (d, 0.4H), 7.68˜7.61 (m, 2H), 7.45˜7.26 (m, 10H), 6.94 (t,1H), 6.79 (s, 1H), 4.81 (s, 1.2H), 4.62 (s, 0.8H), 4.35 (s, 0.8H), 4.13(q, 0.8H), 4.06 (s, 1.2H), 4.03 (q, 1.2H), 3.59 (t, 1.2H), 3.44-3.39 (m,2H), 3.21 (t, 0.8H), 1.19 (t, 1.2H), 1.13 (t, 1.8H).

Example 22N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester

The title compound (2.86 g, 87%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]-acetic acid(1.67 g, 5 mmol) as per the procedure of Example 20. ¹HNMR (500 MHz;DMSO-d₆) δ 10.71 (s, 1H), 8.26 (dd, 1H), 8.16 (dd, 1H), 7.90 (d, 0.6H),7.82 (d, 0.4H), 7.68˜7.60 (m, 2H), 7.34 (d, 2H), 7.00 (t, 1H), 6.93 (d,2H), 5.10 (s, 2H); 4.82 (s, 1.2H), 4.61 (s, 0.8H), 4.34 (s, 0.8H), 4.13(q, 0.8H), 4.05 (s, 1.2H), 4.03 (q, 1.2H), 3.74 (s, 3H), 3.56 (t, 1.2H),3.40˜3.30 (m, 2H), 3.19 (t, 0.8H), 1.19 (t, 1.2H), 1.12 (t, 1.8H).

Example 23N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester

The title compound (5.31 g, 81%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (3.26 g,9.5 mmol) and [4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-aceticacid (3.28 g, 9.03 mmol) as per the procedure of Example 20. ¹H NMR (500MHz; DMSO-d₆) δ 10.72 (s, 1H), 8.27 (dd, 1H), 8.17 (dd, 1H), 7.90 (d,0.6H), 7.82 (d, 0.4H), 7.69˜7.62 (m, 2H), 7.02 (s, 1H), 7.00 (t, 1H),6.94 (s, 2H), 5.09 (s, 2H), 4.80 (s, 1.2H), 4.61 (s, 0.8H), 4.34 (s,0.8H), 4.13 (q, 0.8H), 4.05 (s, 1.2H), 4.03 (q, 1.2H), 3.75 (s, 3H),3.74 (s, 3H), 3.58 (t, 1.2H), 3.42-3.37 (m, 2H), 3.20 (t, 0.8H), 1.20(t, 1.2H), 1.14 (t, 1.8H).

Example 24N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester

The title compound (2.89 g, 86%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetic acid (1.74g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 10.65 (s, 1H), 8.80 (brs, 0.6H), 8.68 (brs, 0.4H), 8.19 (d, 1H), 8.11(d, 1H), 7.81 (d, 0.6H), 7.75 (d, 0.4H), 7.62-7.54 (m, 2H), 6.92 (t,1H), 6.90 (s, 1H), 6.82 (s, 2H), 5.93 (s, 2H), 5.00 (s, 2H), 4.72 (s,1.2H), 4.54 (s, 0.8H), 4.26 (s, 0.8H), 4.01 (q, 0.8H), 4.00 (s, 1.2H),4.96 (q, 1.2H), 3.51 (t, 1.2H), 3.35˜3.30 (m, 2H), 3.13 (t, 0.8H), 1.12,(t, 1.2H), 1.06 (t, 1.8H).

Example 25N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester

The title compound (2.60 g, 85%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetic acid(1.44 g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz;DMSO-d₆) δ 8.89 (t, 0.6H) 8.75 (t, 0.4H) 8.28 (dd, 1H) 8.19 (dd, 1H)7.90 (d, 0.6H) 7.84 (d, 0.4H) 7.67 (m, 2H) 6.99 (m, 1H) 4.81 (s, 1.2H)4.62 (s, 0.8H) 4.35 (s, 0.8H) 4.26 (t, 2H) 4.14 (q, 0.8H) 4.06 (s, 1.2H)4.04 (q, 1.2H) 3.59 (t, 1.2H) 3.42 (m, 2.0H) 3.21 (q, 0.8H) 2.73 (t, 2H)2.12 (s, 3H) 1.20 (t, 1.2H) 1.15 (t, 1.8H).

Example 26N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.91 g, 80%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 m mol) and [6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetic acid (2.02g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 10.93 (s, 1H), 9.00 (s, 0.6H), 8.60 (s, 0.4H), 8.59 (s, 0.414), 8.54(s, 0.6H), 8.33 (d, 1H), 8.28 (m, 1H), 8.19 (d, 1H), 7.69 (m, 2H),7.52-7.29 (m, 10H), 6.83 (s, 1H), 5.47 (s, 1.2H), 5.37 (s, 0.8H), 4.47(s, 0.8H), 4.18 (q, 0.8H), 4.08 (s, 1.2H), 4.03 (q, 1.2H), 3.71 (t,1.2H), 3.61 (q, 1.2H) 3.49 (t, 0.8H) 3.42 (q, 0.8H), 1.24 (t, 1.2H),1.13 (t, 1.8H).

Example 27N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester

To the mixture of N-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycineethyl ester (172 mg, 0.5 mmol),[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetic acid (179 mg, 0.5mmol), and HBTU (190 mg, 0.5 mmol) in DMF (2 ml) was addeddiisopropylethylamine (0.09 ml, 0.5 mmol) at ambient temperature. Theresulting reaction mixture was stirred for 2 h at the same temperatureand 1N HCl was added to the reaction mixture. The resulting precipitateproduct was filtered off, washed with water, and dried in vacuo to givethe title compound as a white solid (0.35 g, 97%). ¹H NMR (500 MHz;DMSO-d₆) δ 8.97 (t, 0.611) 8.76 (t, 0.4H) 8.64 (s, 0.4H) 8.60 (s, 0.6H)8.53 (d, 1H) 8.28 (m, 1H) 8.17 (d, 1H) 7.66 (m, 2H) 7.40 (d, 2H) 6.95(d, 2H) 5.41 (s, 1.2H), 5.20 (s, 0.8H) 5.18 (s, 2H) 4.46 (s, 0.8H) 4.18(q, 0.8H) 4.08 (s, 1.2H) 4.03 (q, 1.2H) 3.75 (s, 3H) 3.70 (q, 1.2H) 3.48(q, 1.2H) 3.42 (t, 0.8H) 3.22 (q, 0.8H) 1.24 (t, 1.2H) 1.13 (t, 1.8H)

Example 28N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester

The title compound (6.28 g, 96%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (3.26 g,9.5 mmol) and [6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-aceticacid (3.50 g, 9.03 mmol) as per the procedure of Example 27.11H NMR (500MHz; DMSO-d₆) δ 10.58 (s, 1H), 8.98 (brs, 0.6H), 8.77 (brs, 0.41H), 8.57(s, 0.4H), 8.53 (s, 0.6H), 8.29 (d, 1H), 8.27 (m, 1H), 8.17 (d, 1H),7.65 (m, 2H), 7.08 (s, 1H), 6.98 (d, 1H), 6.94 (d, 1H), 5.35 (s, 1.2H),5.15 (s, 0.8H), 5.12 (s, 2H), 4.45 (s, 0.8H), 4.18 (q, 0.8H); 4.07 (s,1.2H), 4.01 (q, 1.2H), 3.75 (s, 3H), 3.74 (s, 3H), 3.70 (t, 1.2H), 3.48(t, 1.2H), 3.42 (t, 0.8H), 3.22 (t, 0.8H), 1.23 (t, 1.2H), 1.12 (t,1.8H).

Example 29N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.96 g, 85%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetic acid (1.86g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 10.63 (s, 1H), 8.98 (brs, 0.6H), 8.77 (brs, 0.4H), 8.57 (s, 0.4H),8.52 (s, 0.6H), 8.29 (d, 1H), 8.27 (m, 1H), 8.17 (d, 1H), 7.65 (m, 2H),7.04 (s, 1H), 6.94 (d, 1H), 6.90 (d, 1H), 6.01 (s, 2H), 5.36 (s, 1.2H),5.15 (s, 0.8H), 5.10 (s, 2H), 4.45 (s, 0.811), 4.17 (q, 0.8H), 4.07 (s,1.2H), 4.01 (q, 1.2H), 3.70 (t, 1.2H), 3.48 (t, 1.2H), 3.41 (t, 0.8H),3.22 (t, 0.8H), 1.23 (t, 1.2H), 1.12 (t, 1.8H).

Example 30N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(2-methylthioethoxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.77 g, 85%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [6-N-(2-methylthioethoxycarbonyl)-adenin-9-yl]-acetic acid(1.56 g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz;DMSO-d₆) δ 10.59 (s, 1H), 8.97 (brs, 0.6H), 8.76 (brs, 0.4H), 8.57 (s,0.4H), 8.52 (s, 0.6H), 8.28 (d, 1H), 8.17 (d, 1H), 7.65 (m, 2H), 5.35(s, 1.2H), 5.14 (s, 0.8H), 4.46 (s, 0.8H), 4.17 (q, 0.8H), 4.06 (s,1.2H), 4.02 (q, 1.2H), 3.70 (t, 1.2H), 3.47 (t, 1.2H), 3.42 (t, 0.8H),3.21 (t, 0.8H), 2.12 (s, 3H), 1.23 (t, 1.2H), 1.12 (t, 1.8H).

Example 31N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.61 g, 70%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetic acid (2.10g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 11.63 (bs, 0.1H), 11.23 (bs, 1H), 8.27 (d, 1H), 8.17 (t, 1H), 7.80 (s,0.6H), 7.76 (s, 0.4H), 7.65 (m, 2H), 7.50˜7.25 (m, 10H), 6.86 (s, 1H),5.12 (s, 1.2H), 4.93 (s, 0.8H), 4.44 (s, 0.8H), 4.18 (q, 0.8H), 4.08 (s,1.2H), 4.04 (q, 1.2H), 3.65 (t, 1.2H), 3.45 (t, 1.2H), 3.41 (t, 0.8H),3.21 (t, 0.8H), 1.23 (t, 1.2H), 1.17 (t, 1.8H).

Example 32N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.72 g, 78%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetic acid(1.89 g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz;DMSO-d₆) δ 8.26 (dd, 1H), 8.15 (dd, 1H), 7.80 (s, 0.6H), 7.76 (s, 0.4H),7.65 (m, 2H), 7.36 (d, 2H), 6.94 (d, 2H), 5.16 (s, 2H), 5.09 (s, 1.2H),4.90 (s, 0.8H), 4.42 (s, 0.8H), 4.16 (q, 0.8H), 4.06 (s, 1.2H), 4.03 (q,1.2H), 3.75 (s, 3H), 3.63 (t, 1.2H), 3.43 (t, 1.2H), 3.39 (t, 0.8H),3.20 (t, 0.8H), 1.21 (t, 1.2H), 1.12 (t, 1.8H).

Example 33N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.48 g, 68%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [2-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-aceticacid (2.02 g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500MHz; DMSO-d₆) δ 8.26 (dd, 1H), 8.14 (dd, 1H), 7.80 (s, 0.6H), 7.76 (s,0.4H), 7.65 (m, 2H), 7.04 (s, 1H), 6.95 (m, 2H), 5.15 (s, 2H), 5.09 (s,1.2H), 4.90 (s, 0.8H), 4.42 (s, 0.8H), 4.16 (q, 0.8H), 4.06 (s, 1.2H),4.03 (q, 1.2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.62 (t, 1.2H), 3.43 (t,1.2H), 3.39 (t, 0.8H), 3.20 (t, 0.8H), 1.21 (t, 1.2H), 1.13 (t, 1.8H).

Example 34N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(piperonylyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.71 g, 76%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [2-N-(piperonyloxycarbonyl)-guanin-9-yl]-acetic acid (1.94g, 5 mmol) as per the procedure of Example 20. ¹H NMR (500 MHz; DMSO-d₆)δ 8.27 (d, 1H), 8.15 (dd, 1H), 7.80 (s, 0.6H), 7.76 (s, 0.4H), 7.65 (m,2H), 7.01 (s, 1H), 6.95 (s, 2H), 6.01 (s, 2H), 5.13 (s, 2H), 5.10 (s,1.2H), 4.91 (s, 0.8H), 4.43 (s, 0.8H), 4.16 (q, 0.8H), 4.06 (s, 1.2H),4.03 (q, 1.2H), 3.62 (t, 1.2H), 3.43 (t, 1.2H), 3.39 (t, 0.8H), 3.19 (t,0.8H), 1.21 (t, 1.2H), 1.13 (t, 1.81H).

Example 35N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester

The title compound (2.67 g, 75%) was synthesized by the reaction ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-glycine ethyl ester (1.72 g,5 mmol) and [2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetic acid(1.64 g, 5 mmol) as per the procedure of Example 20.

Example 36N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine

N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycineethyl ester (5.10 g, 10 mmol) was suspended in tetrahydrofuran (20 mL)and the solution of lithium hydroxide monohydrate (1.03 g, 25 mmol) inwater (20 mL) was added. The reaction mixture was stirred for 1 h. atambient temperature. The aqueous solution was acidified by the dropwiseaddition of 1N HCl at 0° C. The title compound was extracted with ethylacetate (3×10 mL), the combined extracts were dried over magnesiumsulfate and filtered. The filtrate was evaporated to dryness in vacuo toafford the desired product (4.57 g, 95%). ¹H NMR (500 MHz; DMSO-d₆) δ11.30 (s, 0.6H), 11.28 (s, 0.4H), 8.88 (s, 0.6H), 8.77 (s, 0.4H), 8.27(d, 1H), 8.18 (d, 1H), 7.69-7.64 (m, 2H), 7.31 (s, 0.6H), 7.23 (s,0.4H), 4.64 (s, 1.2H), 4.45 (s, 0.8H), 4.21 (s, 0.8H), 3.98 (s, 1.2H),3.52 (t, 1.2H), 3.38 (s, 2H), 3.21 (t, 0.8H), 1.73 (s, 3H).

Example 37N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-glycine

The title compound (629 mg, 93%) was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester (705 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 11.00 (brs 1H), 8.90 (brs, 1H), 8.26 (dd, 1H),8.18 (d, 1H), 7.90 (d, 0.6H), 7.82 (d, 0.4H), 7.68-7.61 (m, 2H),7.45˜7.62 (m, 10H), 6.94 (dd, 1H), 6.79 (s, 1H), 4.80 (s, 1.2H), 4.60(s, 0.8H), 4.20 (s, 0.8H), 3.99 (s, 1.2H), 3.56 (t, 1.2H), 3.39 (m, 2H),3.21 (t, 0.8H).

Example 38N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycine

The title compound (605 mg, 96%) was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester (658 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 10.75 (bs, 1H), 8.89 (t, 0.6H), 8.76 (t, 0.4H),8.27 (d, 1H), 8.19 (d, 1H), 7.90 (d, 0.6H), 7.83 (d, 0.4H), 7.65 (m,2H), 7.34 (d, 2H), 7.00 (dd, 1H), 6.92 (d, 2H), 5.10 (s, 2H), 4.80 (s,1.2H), 4.60 (s, 0.8H), 4.25 (s, 0.8H), 4.00 (s, 1.2H), 3.77 (s, 3H),3.56 (t, 1.2H), 3.39 (m, 2H), 3.21 (t, 0.8H).

Example 39N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycine

The title compound (607 mg, 92%) was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester (689 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 10.85 (brs, 1H), 8.88 (t, 0.6H), 8.75 (t,0.4H), 8.28 (d, 1H), 8.19 (d, 1H), 7.90 (d, 0.6H), 7.83 (d, 0.4H), 7.66(m, 2H), 7.03 (s, 1H), 7.01 (dd, 1H), 6.93 (s, 2H), 5.09 (s, 2H), 4.79(s, 1.2H), 4.60 (s, 0.8H), 4.25 (s, 0.8H), 3.99 (s, 1.2H), 3.75 (s, 3H),3.74 (s, 3H), 3.56 (t, 1.2H), 3.40 (m, 2H), 3.20 (dd, 0.8H).

Example 40N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycine

The title compound (632 mg, 92%) was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester (673 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 10.72 (brs, 1H), 8.25 (d, 1H), 8.18 (d, 1H),7.89 (d, 0.4H), 7.82 (d, 0.6H), 7.63 (m, 2H), 6.98 (s, 1H), 6.97° (dd,1H), 6.90 (s, 2H), 6.00 (s, 2H), 5.06 (s, 2H), 4.77 (s, 0.8H), 4.54 (s,1.2H), 3.90 (s, 2H), 3.58 (t, 1.2H), 3.40 (m, 1.6H), 3.20 (t, 1.2H).

Example 41N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetyl}-glycine

The title compound (537 mg, 92%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetyl}-glycineethyl ester (613 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 8.87 (t, 0.6H), 8.74 (t, 0.4H), 8.28 (dd, 1H),8.19 (m, 1H), 7.90 (d, 0.6H), 7.83 (d, 0.4H), 7.66 (m, 2H), 6.98 (m,1H), 4.80 (s, 1.2H), 4.61 (s, 0.8H), 4.26 (t, 3H), 4.25 (s, 0.8H), 4.00(q, 1.2H), 3.57 (t, 1.2H), 3.40 (m, 2.0H), 3.22 (q, 0.8H), 2.73 (t, 2H),2.12 (s, 3H).

Example 42N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetyl}-glycine

The title compound (631 mg, 90%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester (729 mg, 1.0 mmol) as per the procedure of Example 36.¹H-NMR (500 MHz; DMSO-d₆) δ 11.00 (s, 1H), 9.00 (br, 1H), 8.55 (d, 1H),8.32 (d, 1H), 8.26 (m, 1H), 8.17 (m, 1H), 7.64 (m, 2H), 7.52-7.26 (m,10H), 6.81 (s, 1H), 5.35 (s, 1H), 5.13 (s, 1H), 4.33 (s, 1H), 4.00 (s,1H), 3.67 (t, 1H), 3.48 (t, 1H), 3.40 (t, 1H), 3.22 (t, 1H).

Example 43N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycine

The title compound (596 mg, 91%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester (683 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 11.75 (brs, 1H), 8.57 (d, 1H), 8.37 (d, 1H),8.27 (d, 1H), 8.18 (dd, 1H), 7.65 (m, 2H), 7.39 (d, 2H), 6.94 (d, 2H),5.36 (s, 1H), 5.15 (s, 3H), 4.37 (s, 1H), 4.01 (s, 1H), 3.75 (s, 3H),3.68 (t, 1H), 3.48 (q, 1H), 3.40 (q, 1H), 3.22 (q, 1H).

Example 44N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycine

The title compound (597 mg, 87%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester (713 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 11.60 (brs, 1H), 8.56 (s, 0.5H), 8.53 (s,0.5H), 8.29 (d, 1H), 8.27 (d, 1H), 8.18 (dd, 1H), 7.64 (m, 2H), 7.08 (s,1H), 6.98 (d, 1H), 6.94 (d, 1H), 5.34 (s, 1H), 5.13 (s, 1H), 5.12 (s,2H), 4.36 (s, 1H), 4.00 (s, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.67 (t,1H), 3.48 (q, 1H), 3.40 (q, 1H), 3.22 (q, 1H).

Example 45N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetyl}-glycine

The title compound (629 mg, 94%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester (697 mg, 1.0 mmol) as per the procedure of Example 36. ¹HNMR (500 MHz; DMSO-d₆) δ 11.65 (brs, 1H), 8.55 (s, 0.51H), 8.53 (s,0.51H), 8.30 (s, 0.511), 8.27 (s, 0.5H), 8.25 (d, 1H), 8.17 (dd, 1H),7.64 (m, 2H), 7.04 (s, 1H), 6.94 (d, 1H), 6.90 (d, 1H), 6.01 (s, 2H),5.34 (s, 1H), 5.09 (s, 3H), 4.20 (s, 1H), 3.98 (s, 1H), 3.67 (t, 1H),3.49 (t, 1H), 3.41 (t, 1H), 3.23 (t, 1H).

Example 46N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(2-methylthioethoxycarbonyl)-adenin-9-yl]-acetyl}-glycine

The title compound (560 mg, 92%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(2-methylthioethoxycarbonyl)-adenin-9-yl]-acetyl}-glycineethyl ester (637 mg, 1.0 mmol) as per the procedure of Example 36.

Example 47N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-glycine

The title compound (645 mg, 90%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester (745 mg, 1 mmol) as per the procedure of Example 36.1H NMR(500 MHz; DMSO-d₆) δ 11.68 (d, 1H), 11.26 (brs, 2H), 8.94 (t, 0.6H),8.76 (t, 0.4H), 8.27 (d, 114), 8.17 (m, 1H), 7.90 (s, 0.6H), 7.87 (s,0.4H), 7.65 (m, 2H), 7.50-7.25 (m, 10H), 6.86 (s, 1H), 5.12 (s, 1.2H),4.94 (s, 0.8H), 4.35 (s, 0.8H), 4.00 (s, 1.2H), 3.62 (m, 1.2H), 3.40 (m,2H), 3.22 (m, 0.8H).

Example 48N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycine

The title compound (643 mg, 92%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester (699 mg, 1 mmol) as per the procedure of Example 36.1H NMR(500 MHz; DMSO-d₆) δ 8.92 (t, 0.6H), 8.76 (t, 0.4H), 8.26 (dd, 1H), 8.16(dd, 1H), 7.80 (s, 0.6H), 7.75 (s, 0.4H), 7.67-7.61 (m, 2H), 7.36 (d,2H), 6.94 (d, 2H), 5.16 (s, 2H), 5.08 (s, 1.2H), 4.89 (s, 0.8H), 4.33(s, 0.8H), 4.00 (s, 1.2H), 3.74 (s, 3H), 3.60 (t, 1.2H), 3.43 (t, 0.8H),3.38 (m, 1.2H), 3.22 (m, 0.8H).

Example 49N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycine

The title compound (603 mg, 86%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester (729 mg, 1 mmol) as per the procedure of Example 36. ¹H NMR(500 MHz; DMSO-d₆) δ 11.36 (s, 0.5H), 11.34 (s, 0.5H), 8.26 (d, 1H),8.16 (dd, 1H), 7.80 (s, 0.514), 7.75 (s, 0.5H), 7.67-7.61 (m, 2H), 7.03(s, 1H), 6.97-6.94 (m, 2H), 5.15 (s, 2H), 5.08 (s, 1H), 4.88 (s, 1H),4.26 (s, 1H), 3.98 (s, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.60 (t, 1H),3.43 (t, 1H), 3.38 (t, 1H), 3.21 (t, 1H).

Example 50N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(piperonyloxycarbonyl)-guanin-9-yl]-acetyl}-glycine

The title compound (630 mg, 92%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(piperonyloxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester (713 mg, 1 mmol) as per the procedure of Example 36. ¹H NMR(500 MHz; DMSO-d₆) δ 11.36 (s, 0.5H), 11.35 (s, 0.5H), 8.25 (d, 1H),8.16 (dd, 1H), 7.81 (s, 0.5H), 7.76 (s, 0.5H), 7.68-7.61 (m, 2H), 7.00(s, 1H), 6.91 (s, 2H), 6.01 (s, 2H), 5.12 (s, 2H), 5.09 (s, 1H), 4.89(s, 1H), 4.25 (s, 1H), 3.99 (s, 1H), 3.61 (t, 1H), 3.44 (t, 1H), 3.39(t, 1H), 3.21 (t, 1H).

Example 51N-[2-(Benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetyl}-glycine

The title compound (581 mg, 93%) was synthesized from ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetyl}-glycineethyl ester (653 mg, 1 mmol) as per the procedure of Example 36. ¹H NMR(500 MHz; DMSO-d₆) δ 11.35 (s, 0.5H), 11.32 (s, 0.51H), 8.26 (d, 1H),8.16 (dd, 1H), 7.82 (s, 0.5H), 7.79 (s, 0.5H), 7.67-7.61 (m, 2H), 5.08(s, 1H), 4.90 (s, 1H), 4.33 (s, 1H), 4.30 (t, 2H), 3.99 (s, 1H), 3.62(t, 1H), 3.45 (t, 1H), 3.39 (t, 1H), 3.20 (t, 1H), 3.13 (t, 2H), 2.51(s, 3H).

Example 521-(Benzothiazole-2-sulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

To the mixture of 1-(benzothiazole-2-sulfonyl)-piperazin-2-onetrifluoroacetic acid salt (0.83 g, 2.5 mmol), (thymin-1-yl)-acetic acid(0.46 g, 2.5 mmol), and PyBOP (1.43 g, 2.75 mmol) in DMF (12 mL) wasadded diisopropylethylamine (0.61 ml, 3.75 mmol) at ambient temperature.The resulting reaction mixture was stirred for 4 h at the sametemperature and the solvent was removed in vacuo. The residue wasdissolved in dichloromethane (20 mL) and the solution was washed withwater, saturated sodium bicarbonate solution, 1N HCl solution, andsaline. The resulting solution was dried over magnesium sulfate andfiltered. The filtrate was evaporated in vacuo. The resulting residuewas recrystallized in dichloromethane-ethyl ether to afford the titlecompound. ¹H NMR 500 MHz; DMSO-d₆) δ 11.30 (d, 1H), 8.34 (d, 1H), 8.26(m, 1H), 7.71 (m, 2H), 7.34 (s, 0.6H), 7.27 (s, 0.4H), 4.67 (s, 1.2H),4.57 (s, 0.8H), 4.42 (s, 0.8H), 4.27 (s, 1.2H), 4.21 (t, 1.2H), 4.07 (t,0.8H), 3.95 (t, 1.2H), 3.85 (t, 0.8H), 1.74 (s, 3H).

Example 531-(4-Chloro-5-methoxy-benzothiazole-2-sulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

The title compound was synthesized by the reaction of1-(4-chloro-5-methoxy-benzothiazole-2-sulfonyl)-piperazin-2-onetrifluoroacetic acid salt with[4-N-(benzyloxycarbonyl)-cytosin-1-yl]-acetic acid as per the procedureof Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.30 (s, 0.6H), 11.29 (s,0.4H), 8.27 (d, 1H), 7.66 (d, 1H), 7.33 (s, 0.6H), 7.27 (s, 0.4H), 4.67(s, 1.2H), 4.57 (s, 0.8H), 4.42 (s, 0.8H), 4.28 (s, 1.2H), 4.21 (t,1.2H), 4.07 (t, 0.8H), 3.99 (s, 3H), 3.95 (t, 1.2H), 3.85 (t, 0.8H).

Example 541-(Benzothiazole-2-sulfonyl)-4-{[4-N-(benzyloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(benzyloxycarbonyl)-cytosin-1-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.79 (brs, 1H),8.34 (m, 1H), 8.26 (m, 1H), 7.91 (d, 0.6H), 7.85 (d, 0.4H), 7.45˜7.30(m, 5H), 7.10 (t, 1H), 5.18 (s, 2H), 4.82 (s, 1.2H), 4.72 (s, 0.8H),4.45 (s, 0.8H), 4.28 (s, 1.2H), 4.22 (t, 1.2H), 4.07 (t, 0.8H), 3.99 (t,1.2H), 3.86 (t, 0.8H).

Example 551-(Benzothiazole-2-sulfonyl)-4-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.00 (brs, 1H),8.34 (m, 1H), 8.26 (m, 1H), 7.90 (d, 0.6H), 7.84 (d, 0.4H), 7.72 (m,2H), 7.44 (d, 4H), 7.37 (t, 4H), 7.29 (t, 2H), 6.94 (t, 1H), 6.79 (s,1H), 4.82 (s, 1.2H), 4.72 (s, 0.8H), 4.45 (s, 0.8H), 4.28 (s, 1.2H),4.22 (t, 1.2H), 4.07 (t, 0.8H), 3.99 (t, 1.2H), 3.86 (t, 0.8H).

Example 561-(Benzothiazole-2-sulfonyl)-4-{[4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.70 (bs, 1H),8.34 (m, 1H), 8.26 (m, 1H), 7.90 (d, 0.6H), 7.84 (d, 0.4H), 7.72 (m,2H), 7.34 (d, 2H), 7.01 (t, 1H), 6.93 (d, 2H), 5.10 (s, 2H), 4.81 (s,1.2H), 4.71 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.22 (t, 1.2H),4.07 (t, 0.8H), 3.99 (t, 1.2H), 3.85 (t, 0.8H), 3.74 (s, 3H).

Example 571-(Benzothiazole-2-sulfonyl)-4-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]-acetic acid asper the procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.73 (brs,1H), 8.34 (m, 1H), 8.26 (m, 1H), 7.90 (d, 0.6H), 7.84 (d, 0.4H), 7.71(m, 2H), 7.03 (s, 1H), 7.02 (t, 1H), 6.94 (s, 2H), 5.09 (s, 2H), 4.82(s, 1.2H), 4.72 (s, 0.8H), 4.45 (s, 0.8H), 4.27 (s, 1.2H), 4.21 (t,1.2H), 4.06 (t, 0.8H), 3.99 (t, 1.2H), 3.85 (t, 0.8H), 3.75 (s, 3H),3.74 (s, 3H).

Example 581-(Benzothiazole-2-sulfonyl)-4-{[4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(piperonyloxycarbonyl)-cytosin-1-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.76 (brs, 1H),8.36 (m, 1H), 8.28 (m, 1H), 7.92 (d, 0.6H), 7.86 (d, 0.4H), 7.73 (m,2H), 7.03 (t, 1H), 7.00 (s, 1H), 6.92 (s, 2H), 6.03 (s, 2H), 5.08 (s,2H), 4.84 (s, 1.2H), 4.74 (s, 0.8H), 4.47 (s, 0.8H), 4.30 (s, 1.2H),4.24 (t, 1.2H), 4.08 (t, 0.8H), 4.01 (t, 1.2H), 3.89 (t, 0.8H).

Example 591-(Benzothiazole-2-sulfonyl)-4-{[4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]-acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.72 (brs,1H), 8.34 (m, 1H), 8.26 (m, 1H), 7.90 (d, 0.6H), 7.84 (d, 0.4H); 7.72(m, 2H), 7.01 (t, 1H), 4.82 (s, 1.2H), 4.71 (s, 0.8H), 4.45 (s, 0.8H),4.27 (s, 1.2H), 4.26 (t, 2H), 4.22 (t, 1.2H), 4.07 (t, 0.8H), 3.99 (t,1.2H), 3.86 (t, 0.8H), 2.73 (t, 2H), 2.11 (s, 3H).

Example 601-(5-Chloro-benzothiazole-2-sulfonyl)-4-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(5-chloro-benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroaceticacid salt with [4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]acetic acid asper the procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.98 (brs,1H), 8.40 (m, 2H), 7.96 (s, 1H), 7.79 (m, 1H), 7.45 (d, 4H), 7.38 (t,4H), 7.30 (t, 2H), 6.96 (t, 1H), 6.80 (s, 1H), 4.83 (s, 1.2H), 4.72 (s,0.8H), 4.46 (s, 0.8H), 4.28 (s, 1.2H), 4.22 (t, 1.2H), 4.06 (t, 0.8H),3.99 (t, 1.2H), 3.87 (t, 0.8H).

Example 611-(Benzothiazole-2-sulfonyl)-4-{[6-N-(benzyloxycarbonyl)-adenine-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(benzyloxycarbonyl)-adenine-9-yl]-acetic acid as per theprocedure of Example 52. ¹H-NMR (500 MHz; DMSO-d₆) δ 10.81 (brs, 1H),8.60 (d, 1H), 8.36 (m, 1H), 8.28 (m, 2H), 7.73 (m, 2H), 7.46 (d, 2H),7.40 (t, 2H), 7.34 (t, 1H), 5.41 (s, 1.2H), 5.29 (s, 0.8H), 5.23 (s,2H), 4.57 (s, 0.8H), 4.28 (s, 2.4H), 4.09 (m, 2.0H), 3.88 (t, 0.814).

Example 621-(Benzothiazole-2-sulfonyl)-4-{[6-N-(benzhydryloxycarbonyl)-adenine-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(benzhydryloxycarbonyl)-adenine-9-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.15 (brs, 1H),8.60 (d, 1H), 8.45-8.32 (m, 2H), 8.26 (m, 1H), 7.72 (m, 2H), 7.52 (d,4H), 7.38 (t, 4H), 7.29 (t, 2H), 6.83 (s, 1H), 5.41 (s, 1.2H), 5.30 (s,0.8H), 4.57 (s, 0.8H), 4.28 (s, 2.4H), 4.09 (brs, 2.0H), 3.88 (t, 0.8H).

Example 631-(Benzothiazole-2-sulfonyl)-4-{[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(4-methoxybenzyloxycarbonyl)-adenine-9-yl]-acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.60 (bs, 1H),8.59 (d, 1H), 8.34 (m, 2H), 8.27 (m, 1H), 7.72 (m, 2H), 7.38 (d, 2H),6.94 (d, 2H), 5.40 (s, 1.2H), 5.28 (s, 0.8H), 5.14 (s, 2H), 4.56 (s,0.8H), 4.28 (m, 2.4H), 4.09 (t, 2H), 3.87 (t, 0.8H), 3.75 (s, 3M).

Example 641-(Benzothiazole-2-sulfonyl)-4-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenine-9-yl]-acetic acid asper the procedure of Example 52. ¹ NMR (500 MHz; DMSO-d₆) δ 10.58 (s,1H), 8.58 (s, 0.6H), 8.56 (s, 0.4H), 8.35 (m, 1H), 8.30 (s, 0.4H), 8.27(s, 0.6H), 8.26 (m, 1H), 7.73 (m, 2H), 7.07 (s, 1H), 6.98 (d, 1H), 6.94(d, 1H), 5.38 (s, 1.2H), 5.27 (s, 0.8H), 5.12 (s, 2H), 4.56 (s, 0.8H),4.28 (m, 2.4H), 4.09 (t, 2H), 3.87 (t, 0.8H), 3.76 (s, 3H), 3.74 (s,3H).

Example 651-(Benzothiazole-2-sulfonyl)-4-{[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(piperonyloxycarbonyl)-adenine-9-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.64 (s, 1H), 8.58(s, 0.6H), 8.57 (s, 0.4H), 8.34 (m, 1H), 8.31 (s, 0.4H), 8.28 (s, 0.6H),8.27 (m, 1H), 7.72 (m, 2H), 7.04 (s, 1H), 6.94 (d, 1H), 6.90 (d, 1H),6.01 (s, 2H), 5.39 (s, 1.2H), 5.28 (s, 0.8H), 5.09 (s, 2H), 4.57 (s,0.8H), 4.29 (m, 2.4H), 4.09 (t, 2H), 3.88 (t, 0.8H).

Example 661-(Benzothiazole-2-sulfonyl)-4-{[6-N-(2-methylthioethoxycarbonyl)-adenine-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [6-N-(2-methylthioethoxycarbonyl)-adenine-9-yl]-acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 10.80 (brs,1H), 8.61 (d, 1H), 8.42 (s, 0.6H), 8.39 (s, 0.4H), 8.35 (m, 1H), 8.27(m, 1H), 7.73 (m, 2H), 5.42 (s, 1.2H), 5.30 (s, 0.8H), 4.57 (s, 0.8H),4.34-4.25 (m, 4.4H), 4.09 (m, 2.0H), 3.88 (t, 0.8H), 2.79 (t, 2H), 2.13(s, 3H).

Example 671-(Benzothiazole-2-sulfonyl)-4-{[2-N-(benzyloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(benzyloxycarbonyl)-guanin-9-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.35 (bs, 2H),8.34 (m, 1H), 8.25 (m, 1H), 7.81 (s, 0.6H), 7.77 (s, 0.4H), 7.71 (m,2H), 7.45˜7.30 (m, 5H), 5.23 (s, 2H), 5.13 (s, 1.2H), 5.02 (s, 0.8H),4.51 (s, 0.8H), 4.27 (s, 1.2H), 4.24 (t, 1.2H), 4.08 (t, 0.8H), 4.03 (t,1.2H), 3.85 (t, 0.8H).

Example 681-(Benzothiazole-2-sulfonyl)-4-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.62 (bs, H),11.24 (bs, 1H), 8.36 (m, 1H), 8.26 (m, 1H), 7.81 (s, 0.6H), 7.77 (s,0.4H), 7.72 (m, 2H), 7.45 (d, 4H), 7.38 (t, 4H), 7.30 (t, 2H), 6.86 (s,1H), 5.16 (s, 1.2H), 5.05 (s, 0.8H), 4.53 (s, 0.8H), 4.28 (s, 1.2H),4.26 (t, 1.2H), 4.09 (t, 0.8H), 4.05 (t, 1.2H), 3.86 (t, 0.8H).

Example 691-(Benzothiazole-2-sulfonyl)-4-{[2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]-acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.38 (bs, 2H),8.33 (m, 1H), 8.24 (m, 1H), 7.81 (d, 0.6H), 7.77 (d, 0.4H), 7.71 (m,2H), 7.36 (d, 2H), 6.94 (d, 2H), 5.16 (s, 2H), 5.12 (s, 1.2H), 5.02 (s,0.8H), 4.51 (s, 0.8H), 4.26 (s, 1.2H), 4.24 (t, 1.2H), 4.08 (t, 0.8H),4.03 (t, 1.2H), 3.85 (t, 0.8H), 3.75 (s, 3H).

Example 701-(Benzothiazole-2-sulfonyl)-4-{[2-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was, synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]-acetic acid asper the procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.41 (s,1H), 11.37 (s, 1H), 8.34 (m, 1H), 8.24 (m, 1H), 7.81 (s, 0.6H), 7.77 (s,0.4H), 7.71 (m, 2H), 7.04 (s, 1H), 6.95 (d, 1H), 6.94 (d, 1H), 5.15 (s,2H), 5.13 (s, 1.2H), 5.02 (s, 0.8H), 4.51 (s, 0.8H), 4.27 (s, 1.2H),4.24 (t, 1.2H), 4.07 (t, 0.8H), 4.03 (t, 1.2H), 3.84 (t, 0.8H), 3.75 (s,3H), 3.74 (s, 3H).

Example 711-(Benzothiazole-2-sulfonyl)-4-{[2-N-(piperonyloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(piperonyloxycarbonyl)-guanin-9-yl]-acetic acid as per theprocedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.40 (brs, 2H),8.36 (m, 1H), 8.27 (m, 1H), 7.84 (s, 0.6H), 7.78 (s, 0.4H), 7.73 (m,2H), 7.02 (s, 1H), 6.93 (s, 2H), 6.04 (s, 2H), 5.15 (m, 3.2H), 5.05 (s,0.8H), 4.55 (s, 0.8H), 4.30 (s, 1.2H), 4.27 (t, 1.2H), 4.11 (t, 0.8H),4.07 (t, 1.2H), 3.88 (t, 0.8H).

Example 721-(Benzothiazole-2-sulfonyl)-4-{[2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith [2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]-acetic acid as perthe procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.41 (brs,1H), 11.35 (brs, 1H), 8.35 (m, 1H), 8.25 (m, 1H), 7.82 (d, 0.6H), 7.78(d, 0.4H), 7.72 (m, 2H), 5.14 (s, 1.2H), 5.03 (s, 0.8H), 4.51 (s, 0.8H),4.32 (t, 2H), 4.27 (s, 1.2H), 4.24 (t, 1.2H), 4.08 (t, 0.8H), 4.04 (t;1.2H), 3.85 (t, 0.8H), 2.75 (t, 2H), 2.12 (s, 3H).

Example 731-(5-Chloro-benzothiazole-2-sulfonyl)-4-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized by the reaction of1-(5-chloro-benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroaceticacid salt with [2-N-(benzhydryloxycarbonyl)-guanin-9-yl]-acetic acid asper the procedure of Example 52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.62 (brs,H), 11.25 (brs, 1H), 8.42-8.39 (m, 1H), 7.96 (s, 0.6H), 7.82 (s, 0.4H),7.78 (m, 1H), 7.46 (d, 4H), 7.39 (t, 4H), 7.31 (t, 2H), 6.87 (s, 1H),5.17 (s, 1.2H), 5.06 (s, 0.8H), 4.53 (s, 0.8H), 4.29 (s, 1.2H), 4.25 (t,1.2H), 4.08-4.06 (m, 2H), 3.87 (t, 0.8H).

Example 741-(Benzothiazole-2-sulfonyl)-4-[thymin-1-yl]-acetyl-3-methyl-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-3-methyl-piperazin-2-one trifluoroaceticacid salt with (thymin-1-yl)-acetic acid as per the procedure of Example52. ¹H NMR (500 MHz; DMSO-d₆) δ 11.31 (brs, 1H), 8.34 (m, 1H), 8.25 (m,1H), 7.72 (m, 2H), 7.36 (s, 1H), 4.85˜4.60 (m, 2.5H), 4.55˜4.40 (m,0.5H), 4.25˜4.10 (m, 2.5H), 3.85-3.74 (m, 0.5H), 3.50-3.30 (m, 1H), 1.75(s, 3H), 1.44 (d, 1H), 1.29 (d, 2H).

Example 751-(Benzothiazole-2-sulfonyl)-4-[(thymin-1-yl)-acetyl]-piperazin-2-one

Method A

To a solution ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(963 mg, 2 mmole) in tetrahydrofuran (50 mL) was added N-methylmorpholine (3.3 mL, 3 mmole) and then the mixture was cooled to −20° C.After stirring for 5 min at the same temperature, chloroisobutylformate(3.4 mL, 26 mmole) was added to the reaction mixture. The resultingmixture was slowly warmed to 0° C. for 1 h. Then the reaction mixturewas evaporated in vacuo and dissolved in a mixture of ethyl acetate andacetonitrile. The solution was washed with brine and dried over sodiumsulfate and filtered. The filtrate was evaporated in vacuo andtriturated with TETRAHYDROFURAN-ethyl ether (1/1, v/v) to precipitatesolid. The solid was filtered off, washed with ethyl ether, and dried invacuo to give the titled compound (834 mg, 90%).

Method B

The mixture ofN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-[(thymin-1-yl)-acetyl]-glycine(963 mg, 2 mmol) and EDC (460 mg, 2.4 mmol) in DMF (10 mL) was stirredfor 6 h at room temperature. The solvent was removed by evaporation invacuo and the residue was dissolved in dichloromethane (30 mL). Thesolution was washed with 1N HCl solution (20 mL) and water (30 mL). Theorganic layer was dried over MgSO₄, filtered, and concentrated to give ayellow residue. The residue was dissolved in acetone and passed on ashort silica gel. The filtrate was evaporated and the residue wasdissolved in tetrahydrofuran. The organic solution was slowly added toethyl ether to precipitate a white solid, which was collected byfiltration, washed with tetrahydrofuran/ethyl ether (1/2, v/v) and thendried to give the titled compound (862 mg, 93%). ¹H NMR (500 MHz;DMSO-d₆) data are the same as Example 52.

Example 761-(Benzothiazole-2-sulfonyl)-4-{[4-N-(benzhydryloxycarbonyl)-cytosin-1-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(benzhydryloxycarbonyl)]-cytosin-1-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(87%), method B (92%). ¹H NMR-(500 MHz; DMSO-d₆) data are the same asExample 55.

Example 771-(Benzothiazole-2-sulfonyl)-4-{[4-N-(4-methoxybenzyloxycarbonyl)-cytosin-1-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-1-sulfonylamino)-ethyl]-N-{[4-N-(4-methoxybenzyloxycarbonyl)]-cytosin-1-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(85%), method B (90%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 56.

Example 781-(Benzothiazole-2-sulfonyl)-4-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)-cytosin-1-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(3,4-dimethoxybenzyloxycarbonyl)]-cytosin-1-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(85%), method B (90%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 57.

Example 791-(Benzothiazole-2-sulfonyl)-4-{[4-N-(piperonyloxycarbonyl)-cytosin-1-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(piperonyloxycarbonyl)]-cytosin-1-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(91%), method B (95%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 58.

Example 801-(Benzothiazole-2-sulfonyl)-4-{[4-N-(2-methylthioethoxycarbonyl)-cytosin-1-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[4-N-(2-methylthioethoxycarbonyl)]-cytosin-1-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(90%), method B (93%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 59.

Example 811-(Benzothiazole-2-sulfonyl)-4-{[6-N-(benzhydryloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(benzhydryloxycarbonyl)]-adenin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(89%), method B (94%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 62.

Example 821-(Benzothiazole-2-sulfonyl)-4-{[6-N-(4-methoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(4-methoxybenzyloxycarbonyl)]-adenin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(89%), method B (94%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 63.

Example 831-(Benzothiazole-2-sulfonyl)-4-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(3,4-dimethoxybenzyloxycarbonyl)]-adenin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(84%), method B (89%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 64.

Example 841-(Benzothiazole-2-sulfonyl)-4-{[6-N-(piperonyloxycarbonyl)-adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(piperonyloxycarbonyl)]-adenin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(80%), method B (88%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 65.

Example 851-(Benzothiazole-2-sulfonyl)-4-{[6-N-(2-methylthioethoxycarbonyl)adenin-9-yl]-acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[6-N-(2-methylthioethoxycarbonyl)]-adenin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75; Yield: method A(88%), method B (93%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 66.

Example, 861-(Benzothiazole-2-sulfonyl)-4-{[2-N-(benzhydryloxycarbonyl)-guanin-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(benzhydryloxycarbonyl)]-guanin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(90%), method B (92%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 68.

Example 871-(Benzothiazole-2-sulfonyl)-4-{[2-N-(4-methoxybenzyloxycarbonyl)-guanin-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(4-methoxybenzyloxycarbonyl)]-guanin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(86%), method B (88%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 69.

Example 881-(Benzothiazole-2-sulfonyl)-4-{[27-N-(3,4-dimethoxybenzyloxycarbonyl)-guanin-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(3,4-dimethoxybenzyloxycarbonyl)]-guanin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(83%), method B (86%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 70.

Example 891-(Benzothiazole-2-sulfonyl)-4-{[2-N-(piperonyloxycarbonyl)-guanin-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(piperonyloxycarbonyl)]-guanin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(92%), method B (95%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 71.

Example 901-(Benzothiazole-2-sulfonyl)-4-{[2-N-(2-methylthioethoxycarbonyl)-guanin-9-yl]acetyl}-piperazin-2-one

The title compound was synthesized fromN-[2-(benzothiazole-2-sulfonylamino)-ethyl]-N-{[2-N-(2-methylthioethoxycarbonyl)]-guanin-9-yl}-acetyl}-glycineas per the procedures of method A or B of Example 75. Yield: method A(86%), method B (88%). ¹H NMR (500 MHz; DMSO-d₆) data are the same asExample 72.

Example 911-(Benzothiazole-2-sulfonyl)-4-[thymin-1-yl]-acetyl-3-methyl-piperazin-2-one

The title compound was synthesized by the reaction of1-(benzothiazole-2-sulfonyl)-piperazin-2-one trifluoroacetic acid saltwith (thymin-1-yl)-acetic acid as per the procedure of Example 75.Yield: method A (83%), method B (87%). ¹H NMR (500 MHz; DMSO-d₆) dataare the same as Example 74.

Solid Phase Synthesis of PNA Oligomer

General Procedure

PNA oligomer synthesis was conducted manually on a Nova Syn TG aminoresin (Nova biochem., 0.26 mmol/g loading) which is a PEG-graftedpolystyrene resin with amine functionality. The resin was coupled withPAL linker(5-[4-(9-fluorenylmethoxycarbonyl)amino-3,5-dimethoxyphenoxy]pentanoicacid (Advanced ChemTech) by using HBTU as a coupling reagent in DMF. Theresulting resin was treated with 20% piperidine in DMF to activate theamine functional group of PAL linker for PNA oligomer synthesis. The PNAoligomer was synthesized according to the following synthesis cycle. Allreactions and washes of the resin were performed in a fritted vial.

Following is a non-limiting general procedure for solid phase synthesisof PNA oligomer:

-   -   1. Coupling with 10 equiv. of appropriate monomer (20 M in 0.5M        acetic acid in DMF) for 2 h.    -   2. Washing with DMF 3 times.    -   3. Capping with acetic anhydride (5% acetic anhydride and 6%        lutidine in DMF) for 5 mm.    -   4. Washing with DMF 2 times.    -   5. Cleavage over-reacted acetyl group in sulfonamide with        piperidine (1.0 M in DMF) for 5 min.    -   6. Washing with DMF 3 times.    -   7. Deprotection of benzothiazole-2-sulfonyl group with        4-methoxybenzenethiol for 15 min.    -   8. Washing with DMF 3 times

After removal of the final benzothiazole-2-sulfonyl group, the resin iswashed with DMF 3 times and dichloromethane 2 times and dried. Theremoval the protecting group of exocyclic amine and cleavage from theresin was performed in one step by treatment with 25% m-cresol in TFAfor 1.5 h. The resin is filtered off and washed with TFA. Almost TFA ofthe combined filtrate is removed by blowing a steady stream of nitrogen.Then the residue was suspended with ethyl ether and centrifuged. Thesupernatant is carefully decanted off. The residue of crude PNA oligomeris washed one more time by suspension with ethyl ether, centrifuge, andremoval of supernatant. The crude PNA oligomer is analyzed with HPLC andconfirmed by Matrix Assisted Laser Desorption-Time of Flight(MALDI-TOF).

Example 92 Synthesis of PNA oligomer sequence H₂N-CTCGTTTCCA-H (SEQ IDNO: 1)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 17A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 93 Synthesis of PNA oligomer sequence H₂N-TCGTGTCGTA-H (SEQ IDNO:2)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 18A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 94 Synthesis of PNA oligomer sequence H₂N-ACCAGCGGCA-H (SEQ IDNO:3)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 19A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 95 Synthesis of PNA oligomer sequence H₂N-TCTTCTAGTG-H (SEQ IDNO:4)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 20A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 96 Synthesis of PNA oligomer sequence H₂N-GTGCTCCTCC-H (SEQ ID.NO:5)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(C and G) is benzhydryloxycarbonyl. FIGS. 21A-B show (A) HPLC and (B)MALDI-TOF profiles.

Example 97 Synthesis of PNA oligomer sequence H₂N-GTGCATGATG-H (SEQ IDNO:6)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 22A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 98 Synthesis of PNA oligomer sequence H₂N-CCCTACTGTG-H (SEQ IDNO:7)

The title sequence was synthesized manually by the above generalprocedures using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 23A-B show (A) HPLC and(B) MALDI-TOF profiles.

Example 99 Synthesis of PNA oligomer sequence H₂N-CTCATTTCCA-H (SEQ IDNO:8)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A and C) is benzhydryloxycarbonyl. FIGS. 24A-B show (A) HPLC and (B)MALDI-TOF profiles.

Example 100 Synthesis of PNA oligomer sequence H₂N-ACCCTACTGT-H (SEQ IDNO:9)

The title sequence was synthesized manually by the above generalprocedure using monomer of general formula I, wherein R1, R2, R3, R4,and R5 are H and the protecting group of exocyclic amine of nucleobases(A, C, and G) is benzhydryloxycarbonyl. FIGS. 25A-B show, (A) HPLC and(B) MALDI-TOF profiles.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine; experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

1-44. (canceled)
 45. A compound having formula I:

wherein E is nitrogen or C—R′; J is sulfur or oxygen; R′, R1, R2, R3, R4is independently H, halogen, alkyl, nitro, cyano, alkoxy, halogenatedalkyl, halogenated alkoxy, phenyl or halogenated phenyl; R5 is H orprotected or unprotected side chain of natural or unnatural α-aminoacid; and R7 has a formula:

wherein Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₈, Y₉, Y₁ is independently selectedfrom hydrogen, halogen, alkyl, and alkoxy; or

wherein Z₁, Z₂, Z₃, Z₄, Z₅ is independently selected from hydrogen,halogen, alkyl, alkoxy, and methylene dioxy of adjacent two residues; or

wherein R8 is alkyl or phenyl.
 46. The compound of claim 45, wherein Eis nitrogen and J is sulfur.
 47. The compound of claim 45, wherein E isnitrogen and J is oxygen.
 48. The compound of claim 45, wherein E is CHand J is sulfur.
 49. The compound of claim 45, wherein E is CH and J isoxygen.
 50. The compound of claim 45, wherein R5 is H or protected orunprotected side chain of natural α-amino acid.
 51. The compound ofclaim 45, wherein R′ is H, CF₃, F, Cl, Br, I, methyl, phenyl, nitro, orcyano.
 52. The compound of claim 45, wherein R7 has a formula:

wherein Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₈, Y₉, Y₁₀ is independentlyselected from hydrogen, halogen, alkyl, and alkoxy.
 53. The compound ofclaim 45, wherein R7 has a formula:

wherein Z₁, Z₂, Z₃, Z₄, Z₅ is independently selected from hydrogen,halogen, alkyl, alkoxy, and methylene dioxy of adjacent two residues.54. The compound of claim 45, wherein R7 has a formula;

wherein R8 is alkyl or phenyl.
 55. The compound of claim 45, wherein R7is benzyl, benzhydryl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, piperonyl,or 2-methylthioethyl.
 56. The compound of claim 45, wherein R1, R2, R3,and R4 are H.
 57. The compound of claim 45, wherein R2 is Cl, and R1, R3and R4 are H.
 58. The compound of claim 45, wherein R1 is Cl, R2 ismethoxy, and R3, R4 and R5 are H.
 59. The compound of claim 45, whereinR1, R2, R3, and R4 are H; E is nitrogen; and J is sulfur.
 60. A methodof making the compound of claim 45, comprising cyclizing a compound offormula VI in the presence of a coupling reagent that is customarilyused in peptide synthesis or mixed anhydride, wherein the formula VI isrepresented as follows:

wherein E is nitrogen or C—R′; J is sulfur or oxygen; R′, R1, R2, R3, R4is independently H, halogen, alkyl, nitro, cyano, alkoxy, halogenatedalkyl, halogenated alkoxy, phenyl or halogenated phenyl; R5 is H orprotected or unprotected side chain of natural or unnatural α-aminoacid; and R7 has a formula:

wherein Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₈, Y₉, Y₁₀ is independentlyselected from hydrogen, halogen, alkyl, and alkoxy; or

wherein Z₁, Z₂, Z₃, Z₄, Z₅ is independently selected from hydrogen,halogen, alkyl, alkoxy, and methylene dioxy of adjacent two residues; or

wherein R8 is alkyl or phenyl.
 61. A method of making the compound ofclaim 45, comprising coupling reaction of a compound of formula IV witha nucleobase acetic acid moiety in the presence of non-nucleophilicorganic base and a coupling reagent that is customarily used in peptidesynthesis, wherein said formula IV is represented as follows:

wherein E is nitrogen or C—R′; J is sulfur or oxygen; R′, R1, R2, R3, R4is independently H, halogen, alkyl, nitro, cyano, alkoxy, halogenatedalkyl, halogenated alkoxy, phenyl or halogenated phenyl; and R5 is H orprotected or unprotected side chain of natural or unnatural α-aminoacid, wherein said nucleobase acetic acid moiety is represented asfollows:

wherein R7 has a formula:

wherein Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₈, Y₉, Y₁₀ is independentlyselected from hydrogen, halogen, alkyl, and alkoxy; or

wherein Z₁, Z₂, Z₃, Z₄, Z₅ is independently selected from hydrogen,halogen, alkyl, alkoxy, and methylene dioxy of adjacent two residues; or

wherein R8 is alkyl or phenyl.
 62. A compound having the formula:

wherein Q₁, Q₂, Q₃, Q₄, Q₅ is independently H, halogen, NO₂, OH, alkyl,alkoxy, or aryl.
 63. The compound of claim 62, wherein Q₁, Q₂, and Q₃are H; Q₄ is F, Cl, Br, methyl or phenyl; and Q₅ is F, Cl, Br, methyl orphenyl.
 64. The compound of claim 62, wherein Q₁, Q₂, Q₃, isindependently Cl, Br, or NO₂; and Q₄ and Q₅ are H.
 65. The compound ofclaim 62, wherein Q₁, Q₂, Q₃, is independently selected from H, OH,methyl, or methoxy; and Q₄ and Q₅ are H.
 66. The compound of claim 62,wherein Q₁, Q₂, Q₃, Q₄ and Q₅ are H.